Science.gov

Sample records for active site features

  1. Active site mapping of Loxosceles phospholipases D: Biochemical and biological features.

    PubMed

    Vuitika, L; Chaves-Moreira, D; Caruso, I; Lima, M A; Matsubara, F H; Murakami, M T; Takahashi, H K; Toledo, M S; Coronado, M A; Nader, H B; Senff-Ribeiro, A; Chaim, O M; Arni, R K; Veiga, S S

    2016-09-01

    Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D. PMID:27233517

  2. Sterically hindered carboxylate ligands support water-bridged dimetallic centers that model features of metallohydrolase active sites.

    PubMed

    Lee, Dongwhan; Hung, Pei-Lin; Spingler, Bernhard; Lippard, Stephen J

    2002-02-11

    The synthesis and characterization of carboxylate-bridged dimetallic complexes are described. By using m-terphenyl-derived carboxylate ligands, a series of dicobalt(II), dicobalt(III), dinickel(II), and dizinc(II) complexes were synthesized. The compounds are [Co(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (1), [Co(2)(mu-OH(2))(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (2a-c), [Co(2)(mu-OH)(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (3), [Ni(2)(mu-O(2)CAr(Tol))(4)L(2)] (4), [Ni(2)(mu-HO...H)(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (5), and [Zn(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)] (6), where Ar(Tol)CO(2)H = 2,6-di(p-tolyl)benzoic acid and L = pyridine, THF, or N,N-dibenzylethylenediamine. Structural analysis of these complexes revealed that additional bridging ligands can be readily accommodated within the [M(2)(mu-O(2)CAr(Tol))(2)](2+) core, allowing a wide distribution of M...M distances from 2.5745(6) to 4.0169(9) A. Unprecedented bridging units [M(2)(mu-OH(2))(2)(mu-O(2)CR)(2)](n+) and [M(2)(mu-HO...H)(2)(mu-O(2)CR)(2)](n+) were identified in 2a-c and 5, respectively, in which strong hydrogen bonding accommodates shifts of protons from bridging water molecules toward the dangling oxygen atoms of terminal monodentate carboxylate groups. Such a proton shift along the O...H...O coordinate attenuates the donor ability of the anionic carboxylate ligand, which can translate into increased Lewis acidity at the metal centers. Such double activation of bridging water molecules by a Lewis acidic metal center and a metal-bound general base may facilitate the reactivity of metallohydrolases such as methionine aminopeptidase (MAP). PMID:11825079

  3. Prioritizing functional phosphorylation sites based on multiple feature integration

    PubMed Central

    Xiao, Qingyu; Miao, Benpeng; Bi, Jie; Wang, Zhen; Li, Yixue

    2016-01-01

    Protein phosphorylation is an important type of post-translational modification that is involved in a variety of biological activities. Most phosphorylation events occur on serine, threonine and tyrosine residues in eukaryotes. In recent years, many phosphorylation sites have been identified as a result of advances in mass-spectrometric techniques. However, a large percentage of phosphorylation sites may be non-functional. Systematically prioritizing functional sites from a large number of phosphorylation sites will be increasingly important for the study of their biological roles. This study focused on exploring the intrinsic features of functional phosphorylation sites to predict whether a phosphosite is likely to be functional. We found significant differences in the distribution of evolutionary conservation, kinase association, disorder score, and secondary structure between known functional and background phosphorylation datasets. We built four different types of classifiers based on the most representative features and found that their performances were similar. We also prioritized 213,837 human phosphorylation sites from a variety of phosphorylation databases, which will be helpful for subsequent functional studies. All predicted results are available for query and download on our website (Predict Functional Phosphosites, PFP, http://pfp.biosino.org/). PMID:27090940

  4. Activity, selectivity, and stereochemical features in the copper-catalyzed hydrogenative ring-opening of alkyl-substituted cyclopropanes - Nature of active sites

    SciTech Connect

    Palinko, I.; Molnar, A.; Kiss, J.T.; Bartok, M. )

    1990-02-01

    The reactions of four alkyl-substituted cyclopropanes in a hydrogen atmosphere in a closed circulation system have been studied over silica-supported and support-free copper catalysts made by different methods. Both isomerization and direct hydrogen addition occur. The ring opens in both the sterically more hindered and less hindered directions. On the basis of the kinetic data, mechanistic considerations are discussed. The catalysts were studied by N{sub 2}O titration and TPR (temperature-programmed reduction). The properties of the reactions and the TPR data indicate that the active centers are atomic clusters for the direct hydrogenation and Cu(I) ions for the ring-opening from the sterically hindered direction through the formation of olefinic compounds.

  5. Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor

    SciTech Connect

    Bahl, C.; Morisseau, C; Bomberger, J; Stanton, B; Hammock, B; O' Toole, G; Madden, D

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other {alpha}/{beta} hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-{angstrom} resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of {alpha}/{beta} hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.

  6. Supramolecular Chemistry And Self-assembly Special Feature: Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands

    NASA Astrophysics Data System (ADS)

    Hodneland, Christian D.; Lee, Young-Sam; Min, Dal-Hee; Mrksich, Milan

    2002-04-01

    This paper describes a method for the selective and covalent immobilization of proteins to surfaces with control over the density and orientation of the protein. The strategy is based on binding of the serine esterase cutinase to a self-assembled monolayer presenting a phosphonate ligand and the subsequent displacement reaction that covalently binds the ligand to the enzyme active site. Surface plasmon resonance (SPR) spectroscopy showed that cutinase binds irreversibly to a monolayer presenting the capture ligand at a density of 1% mixed among tri(ethylene glycol) groups. The covalent immobilization is specific for cutinase, and the glycol-terminated monolayer effectively prevents unwanted nonspecific adsorption of proteins. To demonstrate that the method could be used to immobilize proteins of interest, a cutinase-calmodulin fusion protein was constructed and immobilized to the monolayer. SPR showed that calcineurin selectively associated with the immobilized calmodulin. This capture ligand immobilization method combines the advantages that the immobilization reaction is highly selective for the intended protein, the tether is covalent and, hence, stable, and the method avoids the need for synthetic modification and rigorous purification of proteins before immobilization. These characteristics make the method well suited to a range of applications and, in particular, for constructing protein microarrays.

  7. Feature selection for splice site prediction: A new method using EDA-based feature ranking

    PubMed Central

    Saeys, Yvan; Degroeve, Sven; Aeyels, Dirk; Rouzé, Pierre; Van de Peer, Yves

    2004-01-01

    Background The identification of relevant biological features in large and complex datasets is an important step towards gaining insight in the processes underlying the data. Other advantages of feature selection include the ability of the classification system to attain good or even better solutions using a restricted subset of features, and a faster classification. Thus, robust methods for fast feature selection are of key importance in extracting knowledge from complex biological data. Results In this paper we present a novel method for feature subset selection applied to splice site prediction, based on estimation of distribution algorithms, a more general framework of genetic algorithms. From the estimated distribution of the algorithm, a feature ranking is derived. Afterwards this ranking is used to iteratively discard features. We apply this technique to the problem of splice site prediction, and show how it can be used to gain insight into the underlying biological process of splicing. Conclusion We show that this technique proves to be more robust than the traditional use of estimation of distribution algorithms for feature selection: instead of returning a single best subset of features (as they normally do) this method provides a dynamical view of the feature selection process, like the traditional sequential wrapper methods. However, the method is faster than the traditional techniques, and scales better to datasets described by a large number of features. PMID:15154966

  8. Aeolin Features and Processes at the Mars Pathfinder Landing Site

    NASA Technical Reports Server (NTRS)

    Greeley, Ronald; Kraft, Michael; Sullivan, Robert; Wilson, Gregory; Bridges, Nathan; Herkenhoff, Ken; Kuzmin, Ruslan; Malin, Michael; Ward, Wes

    1999-01-01

    The Mars Pathfinder landing site contains abundant features attributed to aeolian, or wind, processes. These include wind tails, drift deposits, duneforms of various types, ripplelike features, and ventifacts (the first clearly seen on Mars). Many of these features are consistant with formation involving sand-size particles. Although some features, such as dunes, could develop from saltating sand-size aggregates of finer grains, the discovery of ventifact flutes cut in rocks strongly suggests that at least some of the grains are crystalline, rather than aggregates. Excluding the ventifacts, the orientations of the wind-related features correlate well with the orientations of bright wind steaks seen on Viking Orbiter images in the general area. They also correlate with wind direction predictions from the NASA-Ames General Circulation Model (GCM) which show that the strongest winds in the area occur in the northern hemisphere winter and are directed toward 209 degrees.

  9. Aeolian features and processes at the Mars Pathfinder landing site

    USGS Publications Warehouse

    Greeley, Ronald; Kraft, Michael; Sullivan, Robert; Wilson, Gregory; Bridges, Nathan; Herkenhoff, Ken; Kuzmin, Ruslan O.; Malin, Michael; Ward, Wes

    1999-01-01

    The Mars Pathfinder landing site contains abundant features attributed to aeolian, or wind, processes. These include wind tails, drift deposits, duneforms of various types, ripplelike features, and ventifacts (the first clearly seen on Mars). Many of these features are consistant with formation involving sand-size particles. Although some features, such as dunes, could develop from saltating sand-size aggregates of finer grains, the discovery of ventifact flutes cut in rocks strongly suggests that at least some of the grains are crystalline, rather than aggregates. Excluding the ventifacts, the orientations of the wind-related features correlate well with the orientations of bright wind steaks seen on Viking Orbiter images in the general area. They also correlate with wind direction predictions from the NASA-Ames General Circulation Model (GCM) which show that the strongest winds in the area occur in the northern hemisphere winter and are directed toward 209°. Copyright 1999 by the American Geophysical Union.

  10. Coevolution of active vision and feature selection.

    PubMed

    Floreano, Dario; Kato, Toshifumi; Marocco, Davide; Sauser, Eric

    2004-03-01

    We show that complex visual tasks, such as position- and size-invariant shape recognition and navigation in the environment, can be tackled with simple architectures generated by a coevolutionary process of active vision and feature selection. Behavioral machines equipped with primitive vision systems and direct pathways between visual and motor neurons are evolved while they freely interact with their environments. We describe the application of this methodology in three sets of experiments, namely, shape discrimination, car driving, and robot navigation. We show that these systems develop sensitivity to a number of oriented, retinotopic, visual-feature-oriented edges, corners, height, and a behavioral repertoire to locate, bring, and keep these features in sensitive regions of the vision system, resembling strategies observed in simple insects. PMID:15052484

  11. Site-specific features influence sediment stability of intertidal flats

    NASA Astrophysics Data System (ADS)

    Defew, Emma C.; Tolhurst, Trevor J.; Paterson, David M.

    The factors that influence the sediment stability and the transport of estuarine mudflats are not yet fully understood but knowledge of them is essential in coastal engineering applications and pollution ecology studies. The suggestion that variation in predictive models of sediment stability might be due to site-specific characteristics is investigated using data from four estuarine mudflats (Eden Estuary, Scotland, the Biezelingsche Ham, Zandkreek, and Molenplaat mudflats in The Netherlands). These estuaries differ in their environmental conditions, macrofaunal species composition and local features (e.g. Enteromorpha mats, migratory biofilms). Stable and unstable sediments were compared, and mean chlorophyll-a concentrations and granulometry of the sediments were significantly different between the two groups. Step-wise multiple linear regressions were applied to the sediment stability data of all sites to establish the influences on erosion threshold of microphytobenthic biomass, water content, granulometry, organic carbon content and the abundance of dominant macrofaunal species. The stability of each site was influenced by different factors. Sediment stability of the Eden Estuary was affected by the Enteromorpha bloom; Biezelingsche Ham was influenced by the highly migratory nature of the diatom biofilms and the abundance of Corophium volutator; the polychaete worm Arenicola marina had a net negative effect on sediment stability of the Zandkreek; and the Molenplaat was influenced by microphytobenthic biomass. This research highlights the need for site-specific calibration of models and suggests that a universal proxy parameter for sediment stability is unlikely to be obtained.

  12. Structural and Dynamic Features of F-recruitment Site Driven Substrate Phosphorylation by ERK2.

    PubMed

    Piserchio, Andrea; Ramakrishan, Venkatesh; Wang, Hsin; Kaoud, Tamer S; Arshava, Boris; Dutta, Kaushik; Dalby, Kevin N; Ghose, Ranajeet

    2015-01-01

    The F-recruitment site (FRS) of active ERK2 binds F-site (Phe-x-Phe-Pro) sequences found downstream of the Ser/Thr phospho-acceptor on cellular substrates. Here we apply NMR methods to analyze the interaction between active ERK2 (ppERK2), and a 13-residue F-site-bearing peptide substrate derived from its cellular target, the transcription factor Elk-1. Our results provide detailed insight into previously elusive structural and dynamic features of FRS/F-site interactions and FRS-driven substrate phosphorylation. We show that substrate F-site engagement significantly quenches slow dynamics involving the ppERK2 activation-loop and the FRS. We also demonstrate that the F-site phenylalanines make critical contacts with ppERK2, in contrast to the proline whose cis-trans isomerization has no significant effect on F-site recognition by the kinase FRS. Our results support a mechanism where phosphorylation of the disordered N-terminal phospho-acceptor is facilitated by its increased productive encounters with the ppERK2 active site due to docking of the proximal F-site at the kinase FRS. PMID:26054059

  13. Structural and Dynamic Features of F-recruitment Site Driven Substrate Phosphorylation by ERK2

    PubMed Central

    Piserchio, Andrea; Ramakrishan, Venkatesh; Wang, Hsin; Kaoud, Tamer S.; Arshava, Boris; Dutta, Kaushik; Dalby, Kevin N.; Ghose, Ranajeet

    2015-01-01

    The F-recruitment site (FRS) of active ERK2 binds F-site (Phe-x-Phe-Pro) sequences found downstream of the Ser/Thr phospho-acceptor on cellular substrates. Here we apply NMR methods to analyze the interaction between active ERK2 (ppERK2), and a 13-residue F-site-bearing peptide substrate derived from its cellular target, the transcription factor Elk-1. Our results provide detailed insight into previously elusive structural and dynamic features of FRS/F-site interactions and FRS-driven substrate phosphorylation. We show that substrate F-site engagement significantly quenches slow dynamics involving the ppERK2 activation-loop and the FRS. We also demonstrate that the F-site phenylalanines make critical contacts with ppERK2, in contrast to the proline whose cis-trans isomerization has no significant effect on F-site recognition by the kinase FRS. Our results support a mechanism where phosphorylation of the disordered N-terminal phospho-acceptor is facilitated by its increased productive encounters with the ppERK2 active site due to docking of the proximal F-site at the kinase FRS. PMID:26054059

  14. Salt site performance assessment activities

    SciTech Connect

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

    1983-01-01

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

  15. Perceptions of Business Students' Feature Requirements in Educational Web Sites

    ERIC Educational Resources Information Center

    Hazari, Sunil; Johnson, Barbara

    2007-01-01

    There is paucity of original research that explains phenomena related to content organization and site design of educational Web sites. Educational Web sites are often used to provide Web-based instruction, which itself is a relatively recent phenomenon for business schools, and additional research is needed in this area. Educational Web sites are…

  16. Evaluating Web Sites Featuring Primary Sources on United States History

    ERIC Educational Resources Information Center

    Congleton, Robert J.

    2005-01-01

    Most library Web sites offer lists of recommended Web sites for primary sources with only cursory summaries of the sites. While many of the resources listed are outstanding, too many are dubious in quality, often referring to dead URLs or sites containing no information on their sponsor, source of material, or other information needed to evaluate…

  17. Physicochemical features of the HERG channel drug binding site.

    PubMed

    Fernandez, David; Ghanta, Azad; Kauffman, Gregory W; Sanguinetti, Michael C

    2004-03-12

    Blockade of hERG K(+) channels in the heart is an unintentional side effect of many drugs and can induce cardiac arrhythmia and sudden death. It has become common practice in the past few years to screen compounds for hERG channel activity early during the drug discovery process. Understanding the molecular basis of drug binding to hERG is crucial for the rational design of medications devoid of this activity. We previously identified 2 aromatic residues, Tyr-652 and Phe-656, located in the S6 domain of hERG, as critical sites of interaction with structurally diverse drugs. Here, Tyr-652 and Phe-656 were systematically mutated to different residues to determine how the physicochemical properties of the amino acid side group affected channel block by cisapride, terfenadine, and MK-499. The potency for block by all three drugs was well correlated with measures of hydrophobicity, especially the two-dimensional approximation of the van der Waals hydrophobic surface area of the side chain of residue 656. For residue 652, an aromatic side group was essential for high affinity block, suggesting the importance of a cation-pi interaction between Tyr-652 and the basic tertiary nitrogen of these drugs. hERG also lacks a Pro-Val-Pro motif common to the S6 domain of most other voltage-gated K(+) channels. Introduction of Pro-Val-Pro into hERG reduced sensitivity to drugs but also altered channel gating. Together, these findings assign specific residues to receptor fields predicted by pharmacophore models of hERG channel blockers and provide a refined molecular understanding of the drug binding site. PMID:14699101

  18. OBSERVED ENVIRONMENTAL FEATURES AND THE PHYSICAL ACTIVITY OF ADOLESCENT MALES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: It has recently been reported that adult physical activity was associated with environmental features. The aim of this study was to determine whether environmental features were associated with physical activity among male adolescents. Methods: Physical activity levels of 210 Boy Scouts ...

  19. Modern erosion rates and loss of coastal features and sites, Beaufort Sea coastline, Alaska

    USGS Publications Warehouse

    Jones, Benjamin M.; Hinkel, Kenneth M.; Arp, C.D.; Eisner, Wendy R.

    2008-01-01

    This study presents modern erosion rate measurements based upon vertical aerial photography captured in 1955, 1979, and 2002 for a 100 km segment of the Beaufort Sea coastline. Annual erosion rates from 1955 to 2002 averaged 5.6 m a-1. However, mean erosion rates increased from 5.0 m a-1 in 1955-79 to 6.2 m a-1 in 1979-2002. Furthermore, from the first period to the second, erosion rates increased at 60% (598) of the 992 sites analyzed, decreased at 31% (307), and changed less than ?? 30 cm at 9% (87). Historical observations and quantitative studies over the past 175 years allowed us to place our erosion rate measurements into a longer-term context. Several of the coastal features along this stretch of coastline received Western place names during the Dease and Simpson expedition in 1837, and the majority of those features had been lost by the early 1900s as a result of coastline erosion, suggesting that erosion has been active over at least the historical record. Incorporation of historical and modern observations also allowed us to detect the loss of both cultural and historical sites and modern infrastructure. U.S. Geological Survey topographic maps reveal a number of known cultural and historical sites, as well as sites with modern infrastructure constructed as recently as the 1950s, that had disappeared by the early 2000s as a result of coastal erosion. We were also able to identify sites that are currently being threatened by an encroaching coastline. Our modern erosion rate measurements can potentially be used to predict when a historical site or modern infrastructure will be affected if such erosion rates persist. ?? The Arctic Institute of North America.

  20. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

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

    1990-10-01

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

  1. The Foreign Language Feature Film and Language Teaching Activities.

    ERIC Educational Resources Information Center

    Chappell, Martin

    1986-01-01

    Analysis of foreign language films, featuring consideration of film sequence, image and film analysis, and literary adaptation, is an effective teaching activity with foreign language students. An example illustrates film analysis activities in a first-year French class. (CB)

  2. High Resolution Prediction of Calcium-Binding Sites in 3D Protein Structures Using FEATURE

    PubMed Central

    2015-01-01

    Metal-binding proteins are ubiquitous in biological systems ranging from enzymes to cell surface receptors. Among the various biologically active metal ions, calcium plays a large role in regulating cellular and physiological changes. With the increasing number of high-quality crystal structures of proteins associated with their metal ion ligands, many groups have built models to identify Ca2+ sites in proteins, utilizing information such as structure, geometry, or homology to do the inference. We present a FEATURE-based approach in building such a model and show that our model is able to discriminate between nonsites and calcium-binding sites with a very high precision of more than 98%. We demonstrate the high specificity of our model by applying it to test sets constructed from other ions. We also introduce an algorithm to convert high scoring regions into specific site predictions and demonstrate the usage by scanning a test set of 91 calcium-binding protein structures (190 calcium sites). The algorithm has a recall of more than 93% on the test set with predictions found within 3 Å of the actual sites. PMID:26226489

  3. A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites.

    PubMed

    Beckham, Simone A; Piedrafita, David; Phillips, Carolyn I; Samarawickrema, Nirma; Law, Ruby H P; Smooker, Peter M; Quinsey, Noelene S; Irving, James A; Greenwood, Deanne; Verhelst, Steven H L; Bogyo, Matthew; Turk, Boris; Coetzer, Theresa H; Wijeyewickrema, Lakshmi C; Spithill, Terry W; Pike, Robert N

    2009-07-01

    The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P(2) substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5-7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P(2) position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P(2) Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination. PMID:19401154

  4. Improved coordinates of features in the vicinity of the Viking lander site on Mars

    NASA Technical Reports Server (NTRS)

    Davies, M. E.; Dole, S. H.

    1980-01-01

    The measurement of longitude of the Viking 1 landing site and the accuracy of the coordinates of features in the area around the landing site are discussed. The longitude must be measured photogrammatically from the small crater, Airy 0, which defines the 0 deg meridian on Mars. The computer program, GIANT, which was used to perform the analytical triangulations, and the photogrammetric computation of the longitude of the Viking 1 lander site are described. Improved coordinates of features in the vicinity of the Viking 1 lander site are presented.

  5. Educational Activity Sites for High School Students

    ERIC Educational Resources Information Center

    Troutner, Joanne

    2005-01-01

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

  6. 47 CFR 79.109 - Activating accessibility features.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ACCESSIBILITY OF VIDEO PROGRAMMING Apparatus § 79.109 Activating accessibility features. (a) Requirements... video programming transmitted in digital format simultaneously with sound, including apparatus designed to receive or display video programming transmitted in digital format using Internet protocol,...

  7. Features for voice activity detection: a comparative analysis

    NASA Astrophysics Data System (ADS)

    Graf, Simon; Herbig, Tobias; Buck, Markus; Schmidt, Gerhard

    2015-12-01

    In many speech signal processing applications, voice activity detection (VAD) plays an essential role for separating an audio stream into time intervals that contain speech activity and time intervals where speech is absent. Many features that reflect the presence of speech were introduced in literature. However, to our knowledge, no extensive comparison has been provided yet. In this article, we therefore present a structured overview of several established VAD features that target at different properties of speech. We categorize the features with respect to properties that are exploited, such as power, harmonicity, or modulation, and evaluate the performance of some dedicated features. The importance of temporal context is discussed in relation to latency restrictions imposed by different applications. Our analyses allow for selecting promising VAD features and finding a reasonable trade-off between performance and complexity.

  8. Active transportation safety features around schools in Canada.

    PubMed

    Pinkerton, Bryn; Rosu, Andrei; Janssen, Ian; Pickett, William

    2013-11-01

    The purpose of this study was to describe the presence and quality of active transportation safety features in Canadian school environments that relate to pedestrian and bicycle safety. Variations in these features and associated traffic concerns as perceived by school administrators were examined by geographic status and school type. The study was based on schools that participated in 2009/2010 Health Behaviour in School-aged Children (HBSC) survey. ArcGIS software version 10 and Google Earth were used to assess the presence and quality of ten different active transportation safety features. Findings suggest that there are crosswalks and good sidewalk coverage in the environments surrounding most Canadian schools, but a dearth of bicycle lanes and other traffic calming measures (e.g., speed bumps, traffic chokers). Significant urban/rural inequities exist with a greater prevalence of sidewalk coverage, crosswalks, traffic medians, and speed bumps in urban areas. With the exception of bicycle lanes, the active transportation safety features that were present were generally rated as high quality. Traffic was more of a concern to administrators in urban areas. This study provides novel information about active transportation safety features in Canadian school environments. This information could help guide public health efforts aimed at increasing active transportation levels while simultaneously decreasing active transportation injuries. PMID:24185844

  9. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

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

  10. Improved Species-Specific Lysine Acetylation Site Prediction Based on a Large Variety of Features Set

    PubMed Central

    Wuyun, Qiqige; Zheng, Wei; Zhang, Yanping; Ruan, Jishou; Hu, Gang

    2016-01-01

    Lysine acetylation is a major post-translational modification. It plays a vital role in numerous essential biological processes, such as gene expression and metabolism, and is related to some human diseases. To fully understand the regulatory mechanism of acetylation, identification of acetylation sites is first and most important. However, experimental identification of protein acetylation sites is often time consuming and expensive. Therefore, the alternative computational methods are necessary. Here, we developed a novel tool, KA-predictor, to predict species-specific lysine acetylation sites based on support vector machine (SVM) classifier. We incorporated different types of features and employed an efficient feature selection on each type to form the final optimal feature set for model learning. And our predictor was highly competitive for the majority of species when compared with other methods. Feature contribution analysis indicated that HSE features, which were firstly introduced for lysine acetylation prediction, significantly improved the predictive performance. Particularly, we constructed a high-accurate structure dataset of H.sapiens from PDB to analyze the structural properties around lysine acetylation sites. Our datasets and a user-friendly local tool of KA-predictor can be freely available at http://sourceforge.net/p/ka-predictor. PMID:27183223

  11. Improved Species-Specific Lysine Acetylation Site Prediction Based on a Large Variety of Features Set.

    PubMed

    Wuyun, Qiqige; Zheng, Wei; Zhang, Yanping; Ruan, Jishou; Hu, Gang

    2016-01-01

    Lysine acetylation is a major post-translational modification. It plays a vital role in numerous essential biological processes, such as gene expression and metabolism, and is related to some human diseases. To fully understand the regulatory mechanism of acetylation, identification of acetylation sites is first and most important. However, experimental identification of protein acetylation sites is often time consuming and expensive. Therefore, the alternative computational methods are necessary. Here, we developed a novel tool, KA-predictor, to predict species-specific lysine acetylation sites based on support vector machine (SVM) classifier. We incorporated different types of features and employed an efficient feature selection on each type to form the final optimal feature set for model learning. And our predictor was highly competitive for the majority of species when compared with other methods. Feature contribution analysis indicated that HSE features, which were firstly introduced for lysine acetylation prediction, significantly improved the predictive performance. Particularly, we constructed a high-accurate structure dataset of H.sapiens from PDB to analyze the structural properties around lysine acetylation sites. Our datasets and a user-friendly local tool of KA-predictor can be freely available at http://sourceforge.net/p/ka-predictor. PMID:27183223

  12. WebFEATURE: An interactive web tool for identifying and visualizing functional sites on macromolecular structures.

    PubMed

    Liang, Mike P; Banatao, D Rey; Klein, Teri E; Brutlag, Douglas L; Altman, Russ B

    2003-07-01

    WebFEATURE (http://feature.stanford.edu/webfeature/) is a web-accessible structural analysis tool that allows users to scan query structures for functional sites in both proteins and nucleic acids. WebFEATURE is the public interface to the scanning algorithm of the FEATURE package, a supervised learning algorithm for creating and identifying 3D, physicochemical motifs in molecular structures. Given an input structure or Protein Data Bank identifier (PDB ID), and a statistical model of a functional site, WebFEATURE will return rank-scored 'hits' in 3D space that identify regions in the structure where similar distributions of physicochemical properties occur relative to the site model. Users can visualize and interactively manipulate scored hits and the query structure in web browsers that support the Chime plug-in. Alternatively, results can be downloaded and visualized through other freely available molecular modeling tools, like RasMol, PyMOL and Chimera. A major application of WebFEATURE is in rapid annotation of function to structures in the context of structural genomics. PMID:12824318

  13. Unconscious Semantic Activation Depends on Feature-Specific Attention Allocation

    ERIC Educational Resources Information Center

    Spruyt, Adriaan; De Houwer, Jan; Everaert, Tom; Hermans, Dirk

    2012-01-01

    We examined whether semantic activation by subliminally presented stimuli is dependent upon the extent to which participants assign attention to specific semantic stimulus features and stimulus dimensions. Participants pronounced visible target words that were preceded by briefly presented, masked prime words. Both affective and non-affective…

  14. Masked Primes Activate Feature Representations in Reading Aloud

    ERIC Educational Resources Information Center

    Mousikou, Petroula; Roon, Kevin D.; Rastle, Kathleen

    2015-01-01

    Theories of reading aloud are silent about the role of subphonemic/subsegmental representations in translating print to sound. However, there is empirical evidence suggesting that feature representations are activated in speech production and visual word recognition. In the present study, we sought to determine whether masked primes activate…

  15. Learning Behavior Characterization with Multi-Feature, Hierarchical Activity Sequences

    ERIC Educational Resources Information Center

    Ye, Cheng; Segedy, James R.; Kinnebrew, John S.; Biswas, Gautam

    2015-01-01

    This paper discusses Multi-Feature Hierarchical Sequential Pattern Mining, MFH-SPAM, a novel algorithm that efficiently extracts patterns from students' learning activity sequences. This algorithm extends an existing sequential pattern mining algorithm by dynamically selecting the level of specificity for hierarchically-defined features…

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

    SciTech Connect

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

    2012-02-06

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

  17. Active site specificity of plasmepsin II.

    PubMed Central

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

    1999-01-01

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

  18. Integrated metagenomics and field measurements of polygon features at the NGEE-Arctic Barrow site

    NASA Astrophysics Data System (ADS)

    Tas, N.; Wu, Y.; Smith, L. J.; Ulrich, C.; Kneafsey, T. J.; Torn, M. S.; Hubbard, S. S.; Wullschleger, S. D.; Jansson, J.

    2013-12-01

    Arctic soils contain an estimated 12-42% of terrestrial carbon, most of which is sequestered in permafrost. High latitudes have experienced the greatest regional warming in recent decades and observations suggest that permafrost degradation is now commonly observed in the region. With increasing global temperatures, permafrost soils are becoming a potential source of greenhouse gas (GHG) emissions. Because of widespread permafrost thaw much of the soil organic matter may be available for rapid mineralization by microorganisms in the soil. Yet little is known about the vulnerability of permafrost and the potential response of soil microorganisms to availability of new carbon sources. On the Alaskan North Slope the collapse and rise of soil due to formation of ice wedges and permafrost thaw create distinct features called polygons. As part of the U.S. Department of Energy (DOE) Next Generation Ecosystem Experiment (NGEE) in the Arctic, we aimed to determine the distribution of microbial populations across a range of polygon features and to correlate the microbial data to GHG flux data. To determine the microbial community distribution and metabolic potential, we collected seasonally thawed active layer soil samples along two polygon transects (Site 0 and AB), including high-centered, transitional and low-centered polygons. Illumina HiSeq technology was used to sequence 16SrRNA genes and metagenomes from these active layer soils. The sequence data was correlated to GHG flux measurements and to environmental data from the site, including geophysical and geochemical soil characteristics. Both the microbial communities and the flux measurements varied along the polygon transect. Each polygon had a distinct microbial community structure; however, these microbial communities shared many metabolic capabilities. For example, many genes involved in degradation of chitin could be found all three polygons. Functional genes involved in methanogenesis and CH4-flux measurements

  19. 77 FR 48550 - Technicolor Creative Services, Post Production Feature Mastering Division Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-14

    ... From the Federal Register Online via the Government Publishing Office ] DEPARTMENT OF LABOR Employment and Training Administration Technicolor Creative Services, Post Production Feature Mastering Division Including On-Site Leased Workers From Ajilon Professional Staffing and KForce, Hollywood, CA; Notice of Affirmative Determination...

  20. Features of Digital African American Language in a Social Network Site

    ERIC Educational Resources Information Center

    Cunningham, Jennifer M.

    2014-01-01

    This study examines a social network site (SNS) where specific interlocutors communicate by combining aspects of academic American English (AE), digital language (DL), and African American Language (AAL)--creating a digital form of AAL or digital AAL (DAAL). This article describes the features of DAAL in the discursive, online context of MySpace,…

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

    ERIC Educational Resources Information Center

    Heo, Gyeong Mi; Lee, Romee

    2013-01-01

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

  2. Terrestrial Analogs to Wind-Related Features at the Viking and Pathfinder Landing Sites on Mars

    NASA Technical Reports Server (NTRS)

    Greeley, Ronald; Bridges, Nathan T.; Kuzmin, Ruslan O.; Laity, Julie E.

    2002-01-01

    Features in the Mojave Desert and Iceland provide insight into the characteristics and origin of Martian wind-related landforms seen by the Viking and Pathfinder landers. The terrestrial sites were chosen because they exhibit diverse wind features that are generally well understood. These features have morphologies comparable to those on Mars and include origins by deposition and erosion, with erosional processes modifying both soils and rocks. Duneforms and drifts are the most common depositional features seen at the Martian landing sites and indicate supplies of sand-sized particles blown by generally unidirectional winds. Erosional features include lag deposits, moat-like depressions around some rocks, and exhumed soil horizons. They indicate that wind can deflate at least some sediments and that this process is particularly effective where the wind interacts with rocks. The formation of ripples and wind tails involves a combination of depositional and erosional processes. Rock erosional features, or ventifacts, are recognized by their overall shapes, erosional flutes, and characteristic surface textures resulting from abrasion by windblown particles. The physics of saltation requires that particles in ripples and duneforms are predominantly sand-sized (60-2000 microns). The orientations of duneforms, wind tails, moats, and ventifacts are correlated with surface winds above particle threshold. Such winds are influenced by local topography and are correlated with winds at higher altitudes predicted by atmospheric models.

  3. Application of musical timbre discrimination features to active sonar classification

    NASA Astrophysics Data System (ADS)

    Young, Victor W.; Hines, Paul C.; Pecknold, Sean

    2005-04-01

    In musical acoustics significant effort has been devoted to uncovering the physical basis of timbre perception. Most investigations into timbre rely on multidimensional scaling (MDS), in which different musical sounds are arranged as points in multidimensional space. The Euclidean distance between points corresponds to the perceptual distance between sounds and the multidimensional axes are linked to measurable properties of the sounds. MDS has identified numerous temporal and spectral features believed to be important to timbre perception. There is reason to believe that some of these features may have wider application in the disparate field of underwater acoustics, since anecdotal evidence suggests active sonar returns from metallic objects sound different than natural clutter returns when auralized by human operators. This is particularly encouraging since attempts to develop robust automatic classifiers capable of target-clutter discrimination over a wide range of operational conditions have met with limited success. Spectral features relevant to target-clutter discrimination are believed to include click-pitch and envelope irregularity; relevant temporal features are believed to include duration, sub-band attack/decay time, and time separation pitch. Preliminary results from an investigation into the role of these timbre features in target-clutter discrimination will be presented. [Work supported by NSERC and GDC.

  4. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

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

  5. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

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

  6. PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

    PubMed

    Song, Jiangning; Tan, Hao; Perry, Andrew J; Akutsu, Tatsuya; Webb, Geoffrey I; Whisstock, James C; Pike, Robert N

    2012-01-01

    The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s). Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database) with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate sequence using

  7. ON THE 10 mum SILICATE FEATURE IN ACTIVE GALACTIC NUCLEI

    SciTech Connect

    Nikutta, Robert; Elitzur, Moshe; Lacy, Mark E-mail: moshe@pa.uky.ed

    2009-12-20

    The 10 mum silicate feature observed with Spitzer in active galactic nuclei (AGNs) reveals some puzzling behavior. It (1) has been detected in emission in type 2 sources, (2) shows broad, flat-topped emission peaks shifted toward long wavelengths in several type 1 sources, and (3) is not seen in deep absorption in any source observed so far. We solve all three puzzles with our clumpy dust radiative transfer formalism. Addressing (1), we present the spectral energy distribution (SED) of SST1721+6012, the first type 2 quasar observed to show a clear 10 mum silicate feature in emission. Such emission arises in models of the AGN torus easily when its clumpy nature is taken into account. We constructed a large database of clumpy torus models and performed extensive fitting of the observed SED. We find that the cloud radial distribution varies as r {sup -1.5} and the torus contains 2-4 clouds along radial equatorial rays, each with optical depth at visual approx60-80. The source bolometric luminosity is approx3 x 10{sup 12} L{sub sun}. Our modeling suggests that approx<35% of objects with tori sharing these characteristics and geometry would have their central engines obscured. This relatively low obscuration probability can explain the clear appearance of the 10 mum emission feature in SST1721+6012 together with its rarity among other QSO2. Investigating (2), we also fitted the SED of PG1211+143, one of the first type 1 QSOs with a 10 mum silicate feature detected in emission. Together with other similar sources, this QSO appears to display an unusually broadened feature whose peak is shifted toward longer wavelengths. Although this led to suggestions of non-standard dust chemistry in these sources, our analysis fits such SEDs with standard galactic dust; the apparent peak shifts arise from simple radiative transfer effects. Regarding (3), we find additionally that the distribution of silicate feature strengths among clumpy torus models closely resembles the observed

  8. Building a sense of virtual community: the role of the features of social networking sites.

    PubMed

    Chen, Chi-Wen; Lin, Chiun-Sin

    2014-07-01

    In recent years, social networking sites have received increased attention because of the potential of this medium to transform business by building virtual communities. However, theoretical and empirical studies investigating how specific features of social networking sites contribute to building a sense of virtual community (SOVC)-an important dimension of a successful virtual community-are rare. Furthermore, SOVC scales have been developed, and research on this issue has been called for, but few studies have heeded this call. On the basis of prior literature, this study proposes that perceptions of the three most salient features of social networking sites-system quality (SQ), information quality (IQ), and social information exchange (SIE)-play a key role in fostering SOVC. In particular, SQ is proposed to increase IQ and SIE, and SIE is proposed to enhance IQ, both of which thereafter build SOVC. The research model was examined in the context of Facebook, one of the most popular social networking sites in the world. We adopted Blanchard's scales to measure SOVC. Data gathered using a Web-based questionnaire, and analyzed with partial least squares, were utilized to test the model. The results demonstrate that SIE, SQ, and IQ are the factors that form SOVC. The findings also suggest that SQ plays a fundamental role in supporting SIE and IQ in social networking sites. Implications for theory, practice, and future research directions are discussed. PMID:24690087

  9. Features of the Active Evening Plasma Sheet from MMS

    NASA Astrophysics Data System (ADS)

    Moore, T. E.; Chandler, M. O.; Avanov, L. A.; Burch, J. L.; Coffey, V. N.; Ergun, R. E.; Fuselier, S. A.; Gershman, D. J.; Giles, B. L.; Lavraud, B.; MacDonald, E.; Mauk, B.; Mukai, T.; Nakamura, R.; Pollock, C. J.; Russell, C. T.; Saito, Y.; Sauvaud, J. A.; Torbert, R. B.; Yokota, S.

    2015-12-01

    The Magnetospheric Multiscale (MMS) mission, consisting of four identical plasmas and fields observatories, was launched into a 12 RE elliptical equatorial orbit in March 2015 and was in the process of being commissioned through August 2015. During commissioning, the orbit apogee rotated from near midnight through the evening toward the dusk sector and occasionally captured new observations of the plasma sheet, its boundary layers, and the magnetospheric tail lobes. On 22-23 June, an especially active plasma sheet was involved in a major geospace storm that developed a ring current with 200 nT DST. We report on the ion kinetic and flow features of this active plasma sheet, comparing them with familiar observations from earlier missions, as an exercise in validating the MMS observations and assessing their capabilities to provide higher time resolution in multi-point views of thin, fast-moving structures. The observed features include but are not limited to cold lobal wind streams in the lobes, tailward flowing auroral beams and conics, hot earthward field-aligned flows and counter-flows, fast cross-field convection of some flows toward the neutral sheet, and the hot isotropic plasma sheet proper. Relationships between these features, the ionosphere, and the reconnecting magnetotail will be explored and discussed, seeking preliminary conclusions.

  10. Chemical and structural features influencing the biological activity of curcumin.

    PubMed

    Priyadarsini, K Indira

    2013-01-01

    Curcumin, a polyphenolic natural product, exhibits therapeutic activity against a number of diseases, attributed mainly to its chemical structure and unique physical, chemical, and biological properties. It is a diferuloyl methane molecule [1,7-bis (4-hydroxy-3- methoxyphenyl)-1,6-heptadiene-3,5-dione)] containing two ferulic acid residues joined by a methylene bridge. It has three important functionalities: an aromatic o-methoxy phenolic group, α, β-unsaturated β-diketo moiety and a seven carbon linker. Extensive research in the last two decades has provided evidence for the role of these different functional groups in its crucial biological activities. A few highlights of chemical structural features associated with the biological activity of curcumin are: The o-methoxyphenol group and methylenic hydrogen are responsible for the antioxidant activity of curcumin, and curcumin donates an electron/ hydrogen atom to reactive oxygen species. Curcumin interacts with a number of biomolecules through non-covalent and covalent binding. The hydrogen bonding and hydrophobicity of curcumin, arising from the aromatic and tautomeric structures along with the flexibility of the linker group are responsible for the non-covalent interactions. The α, β-unsaturated β-diketone moiety covalently interacts with protein thiols, through Michael reaction. The β-diketo group forms chelates with transition metals, there by reducing the metal induced toxicity and some of the metal complexes exhibit improved antioxidant activity as enzyme mimics. New analogues with improved activity are being developed with modifications on specific functional groups of curcumin. The physico-chemical and structural features associated with some of the biological activities of curcumin and important analogues are summarized in this article. PMID:23116315

  11. Desired features of smartphone applications promoting physical activity.

    PubMed

    Rabin, Carolyn; Bock, Beth

    2011-12-01

    Approximately one-third of adults in the United States are physically inactive. This is a significant public health concern as physical activity (PA) can influence the risk of cardiovascular disease, diabetes, and certain forms of cancer. To minimize these health risks, effective PA interventions must be developed and disseminated to the vast number of individuals who remain sedentary. Smartphone technology presents an exciting opportunity for delivering PA interventions remotely. Although a number of PA applications are currently available for smartphones, these "apps" are not based on established theories of health behavior change and most do not include evidence-based features (e.g., reinforcement and goal setting). Our aim was to collect formative data to develop a smartphone PA app that is empirically and theoretically-based and incorporates user preferences. We recruited 15 sedentary adults to test three currently available PA smartphone apps and provide qualitative and quantitative feedback. Findings indicate that users have a number of specific preferences with regard to PA app features, including that apps provide automatic tracking of PA (e.g., steps taken and calories burned), track progress toward PA goals, and integrate a music feature. Participants also preferred that PA apps be flexible enough to be used with several types of PA, and have well-documented features and user-friendly interfaces (e.g., a one-click main page). When queried by the researcher, most participants endorsed including goal-setting and problem-solving features. These findings provide a blue print for developing a smartphone PA app that incorporates evidence-based components and user preferences. PMID:22010977

  12. Occurrence of neanderthal features in mandibles from the Atapuerca-SH site.

    PubMed

    Rosas, A

    2001-01-01

    Analysis of variation and distribution of evolutionary novelties is meaningful in understanding evolutionary processes. The mandible, as a morphological complex, comprises a large number of derived Neanderthal features. The present study investigates whether the features usually considered as European lineage apomorphies evolved independently; the occurrence of these features is studied in the mandibles from the Sima de los Huesos (SH) site (Atapuerca, Spain). For comparative purposes, a large sample of Neanderthal mandibles as well as older fossil Homo specimens have been used for the study. Chi-square tests were employed to test for independence. The SH mandibles present a set of features that clearly show the basic architecture of the Neanderthal mandible. A highly significant association is detected in the variation of the position of the mental foramen, the lateral prominence, and the anterior marginal tubercle, as well as in the development of retromolar space. However, a much weaker association is detected in the features of the internal aspect of the mandible, with a few exceptions. Features of the external aspect of the mandible occur chronologically earlier than those observed in the internal aspect. The hypothesis that two distinct and consecutive morphological processes have driven the emergence of the European lineage throughout the Middle Pleistocene is proposed. A first transformation affects the mandible by means of backwards displacement of the structures located at the external aspect, as well as the position of the condyle. A second process would modify the features of the internal aspect of the mandible, in which the relief of the masseteric and pterygoid fossae are affected, in association with a spatial rearrangement of the corpus and ramus. Analyzed individually, some of the considered features may be questioned as Neanderthal apomorphies (Trinkaus,1993; Franciscus and Trinkaus, 995); however, the joint occurrence of many of them suggests

  13. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

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

    1985-01-01

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

  14. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

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

    1992-02-01

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

  15. Active shape models with optimised texture features for radiotherapy

    NASA Astrophysics Data System (ADS)

    Cheng, K.; Montgomery, D.; Yang, F.; McLaren, D. B.; McLaughlin, S.; Nailon, W. H.

    2014-03-01

    There is now considerable interest in radiation oncology on the use of shape models of anatomy to improve target delineation and assess anatomical disparity at time of radiotherapy. In this paper a texture based active shape model (ASM) is presented for automatic delineation of the gross tumor volume (GTV), containing the prostate, on computed tomography (CT) images of prostate cancer patients. The model was trained on two-dimensional (2D) contours identified by a radiation oncologist on sequential CT image slices. A three-dimensional (3D) GTV shape was constructed from these and iteratively aligned using Procrustes analysis. To train the model the shape deformation variance was learnt using the Active Shape Model (ASM) approach. In a novel development to this approach a profile feature was selected from pre-computed texture features by minimizing the Mahalanobis distance to obtain the most distinct feature for each landmark. The interior of the GTV was modelled using quantile histograms to initialize the shape model on new cases. From the archive of 42 cases of contoured CT scans, 32 cases were randomly selected for training the model and 10 cases for evaluating performance. The gold standard was defined by the radiation oncologist. The shape model achieved an overall Dice coefficient of 0.81 for all test cases. Performance was found to increase, mean Dice coefficient of 0.87, when the volume size of the new case was similar to the mean shape of the model. With further work the approach has the potential to be used in real-time delineation of target volumes and improve segmentation accuracy.

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

    PubMed Central

    Elias, Mikael; Chabriere, Eric

    2013-01-01

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

  17. PGlcS: Prediction of protein O-GlcNAcylation sites with multiple features and analysis.

    PubMed

    Zhao, Xiaowei; Ning, Qiao; Chai, Haiting; Ai, Meiyue; Ma, Zhiqiang

    2015-09-01

    As a widespread type of protein post-translational modification, O-GlcNAcylation plays crucial regulatory roles in almost all cellular processes and is related to some diseases. To deeply understand O-GlcNAcylated mechanisms, identification of substrates and specific O-GlcNAcylated sites is crucial. Experimental identification is expensive and time-consuming, so computational prediction of O-GlcNAcylated sites has considerable value. In this work, we developed a novel O-GlcNAcylated sites predictor called PGlcS (Prediction of O-GlcNAcylated Sites) by using k-means cluster to obtain informative and reliable negative samples, and support vector machines classifier combined with a two-step feature selection. The performance of PGlcS was evaluated using an independent testing dataset resulting in a sensitivity of 64.62%, a specificity of 68.4%, an accuracy of 68.37%, and a Matthew׳s correlation coefficient of 0.0697, which demonstrated PGlcS was very promising for predicting O-GlcNAcylated sites. The datasets and source code were available in Supplementary information. PMID:26116363

  18. Active sites in char gasification: Final technical report

    SciTech Connect

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

    1987-09-01

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

  19. Search for DNA conformational features for functional sites. Investigation of the TATA box

    SciTech Connect

    Ponomarenko, M.P.; Ponomarenko, J.V.; Kel, A.E.; Kolchanov, N.A.

    1996-12-31

    A method for searching for DNA conformational features significant for functional sites is developed. The method uses helical angles averaged for known X-ray structures. Nucleotide sequences are assigned mean angles in a given region. Choice of the significant angles is based on their capabilities to discriminate functional sites from random sequences. The yeast, invertebrate, and vertebrate TATA boxes are analyzed using this method. Regions neighboring the TATA boxes are found to have smaller helical twist and roll angles. The results agree with the experimental data on Dickerson-Drew dodecamers. There is a significant decrease in the length of a small roll angle region with increasing complexity of taxon organization. 28 refs., 3 figs., 3 tabs.

  20. Structural Features of a 3′ Splice Site in Influenza A

    PubMed Central

    2015-01-01

    Influenza A is an RNA virus with a genome of eight negative sense segments. Segment 7 mRNA contains a 3′ splice site for alternative splicing to encode the essential M2 protein. On the basis of sequence alignment and chemical mapping experiments, the secondary structure surrounding the 3′ splice site has an internal loop, adenine bulge, and hairpin loop when it is in the hairpin conformation that exposes the 3′ splice site. We report structural features of a three-dimensional model of the hairpin derived from nuclear magnetic resonance spectra and simulated annealing with restrained molecular dynamics. Additional insight was provided by modeling based on 1H chemical shifts. The internal loop containing the 3′ splice site has a dynamic guanosine and a stable imino (cis Watson–Crick/Watson–Crick) GA pair. The adenine bulge also appears to be dynamic with the A either stacked in the stem or forming a base triple with a Watson–Crick GC pair. The hairpin loop is a GAAA tetraloop closed by an AC pair. PMID:25909229

  1. Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features

    PubMed Central

    Cayrou, Christelle; Coulombe, Philippe; Vigneron, Alice; Stanojcic, Slavica; Ganier, Olivier; Peiffer, Isabelle; Rivals, Eric; Puy, Aurore; Laurent-Chabalier, Sabine; Desprat, Romain; Méchali, Marcel

    2011-01-01

    In metazoans, thousands of DNA replication origins (Oris) are activated at each cell cycle. Their genomic organization and their genetic nature remain elusive. Here, we characterized Oris by nascent strand (NS) purification and a genome-wide analysis in Drosophila and mouse cells. We show that in both species most CpG islands (CGI) contain Oris, although methylation is nearly absent in Drosophila, indicating that this epigenetic mark is not crucial for defining the activated origin. Initiation of DNA synthesis starts at the borders of CGI, resulting in a striking bimodal distribution of NS, suggestive of a dual initiation event. Oris contain a unique nucleotide skew around NS peaks, characterized by G/T and C/A overrepresentation at the 5′ and 3′ of Ori sites, respectively. Repeated GC-rich elements were detected, which are good predictors of Oris, suggesting that common sequence features are part of metazoan Oris. In the heterochromatic chromosome 4 of Drosophila, Oris correlated with HP1 binding sites. At the chromosome level, regions rich in Oris are early replicating, whereas Ori-poor regions are late replicating. The genome-wide analysis was coupled with a DNA combing analysis to unravel the organization of Oris. The results indicate that Oris are in a large excess, but their activation does not occur at random. They are organized in groups of site-specific but flexible origins that define replicons, where a single origin is activated in each replicon. This organization provides both site specificity and Ori firing flexibility in each replicon, allowing possible adaptation to environmental cues and cell fates. PMID:21750104

  2. Site-Specific DNA Structural and Dynamic Features Revealed by Nucleotide-Independent Nitroxide Probes

    SciTech Connect

    Popova, Anna; Kalai, Tamas; Hideg, Kalman; Qin, Peter Z.

    2009-09-15

    In site-directed spin labeling, a covalently attached nitroxide probe containing a chemically inert unpaired electron is utilized to obtain information on the local environment of the parent macromolecule. Studies presented here examine the feasibility of probing local DNA structural and dynamic features using a class of nitroxide probes that are linked to chemically substituted phosphorothioate positions at the DNA backbone. Two members of this family, designated as R5 and R5a, were attached to eight different sites of a dodecameric DNA duplex without severely perturbing the native B-form conformation. Measured X-band electron paramagnetic resonance (EPR) spectra, which report on nitroxide rotational motions, were found to vary depending on the location of the label (e.g., duplex center vs termini) and the surrounding DNA sequence. This indicates that R5 and R5a can provide information on the DNA local environment at the level of an individual nucleotide. As these probes can be attached to arbitrary nucleotides within a nucleic acid sequence, they may provide a means to “scan” a given DNA molecule in order to interrogate its local structural and dynamic features.

  3. Modeling place field activity with hierarchical slow feature analysis

    PubMed Central

    Schönfeld, Fabian; Wiskott, Laurenz

    2015-01-01

    What are the computational laws of hippocampal activity? In this paper we argue for the slowness principle as a fundamental processing paradigm behind hippocampal place cell firing. We present six different studies from the experimental literature, performed with real-life rats, that we replicated in computer simulations. Each of the chosen studies allows rodents to develop stable place fields and then examines a distinct property of the established spatial encoding: adaptation to cue relocation and removal; directional dependent firing in the linear track and open field; and morphing and scaling the environment itself. Simulations are based on a hierarchical Slow Feature Analysis (SFA) network topped by a principal component analysis (ICA) output layer. The slowness principle is shown to account for the main findings of the presented experimental studies. The SFA network generates its responses using raw visual input only, which adds to its biological plausibility but requires experiments performed in light conditions. Future iterations of the model will thus have to incorporate additional information, such as path integration and grid cell activity, in order to be able to also replicate studies that take place during darkness. PMID:26052279

  4. [Features of active control used in pharmacological studies].

    PubMed

    Nikol'skaia, K A; Kondashevskaia, M V

    2003-09-01

    The effects of 5 injections of salt solution and unfractionary heparin in dose 0.36 microgram/kg (Serva, Germany 10 kDa, activity 180 U/mg) have been studied in Wistar rats. It was found that two injections of salt solution were enough to form a stable defensive state in all rats which was manifested as an expectation of pain in tail-flick testing. The defensive motivation provoked by the injections negatively influenced the learning process as saline-induced rats refused to solve a food-getting task in a problem situation. Explorative and locomotor activities were depressed in these rats and were accompanied by numerous stressful and neurotic-like manifestations. Unlike saline-rats, practically all heparinized-rats instead of 45% of intact rats were able to solve a cognitive task despite the injections. Anxiety was decreased, but sensitivity to different external factors was increased in the heparin-induced rats. Formed habit in these rats was characterized by a high organization and stability. However, the majority heparin effects in tail-flick test were discovered when comparing the heparin-induced rats with intact ones and were not observed in comparison with the saline-rats. It is suggested that the saline-control should be considered as specific control having defensive features which are necessary to take into account in interpretation of effects of other pharmacological preparations. PMID:14758633

  5. An evaluation of applicability of seismic refraction method in identifying shallow archaeological features A case study at archaeological site

    NASA Astrophysics Data System (ADS)

    Jahangardi, Morteza; Hafezi Moghaddas, Naser; Keivan Hosseini, Sayyed; Garazhian, Omran

    2015-04-01

    We applied the seismic refraction method at archaeological site, Tepe Damghani located in Sabzevar, NE of Iran, in order to determine the structures of archaeological interests. This pre-historical site has special conditions with respect to geographical location and geomorphological setting, so it is an urban archaeological site, and in recent years it has been used as an agricultural field. In spring and summer of 2012, the third season of archaeological excavation was carried out. Test trenches of excavations in this site revealed that cultural layers were often disturbed adversely due to human activities such as farming and road construction in recent years. Conditions of archaeological cultural layers in southern and eastern parts of Tepe are slightly better, for instance, in test trench 3×3 m²1S03, third test trench excavated in the southern part of Tepe, an adobe in situ architectural structure was discovered that likely belongs to cultural features of a complex with 5 graves. After conclusion of the third season of archaeological excavation, all of the test trenches were filled with the same soil of excavated test trenches. Seismic refraction method was applied with12 channels of P geophones in three lines with a geophone interval of 0.5 meter and a 1.5 meter distance between profiles on test trench 1S03. The goal of this operation was evaluation of applicability of seismic method in identification of archaeological features, especially adobe wall structures. Processing of seismic data was done with the seismic software, SiesImager. Results were presented in the form of seismic section for every profile, so that identification of adobe wall structures was achieved hardly. This could be due to that adobe wall had been built with the same materials of the natural surrounding earth. Thus, there is a low contrast and it has an inappropriate effect on seismic processing and identifying of archaeological features. Hence the result could be that application of

  6. Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage sites protect mice from MRSA infection

    PubMed Central

    Schmelcher, Mathias; Shen, Yang; Nelson, Daniel C.; Eugster, Marcel R.; Eichenseher, Fritz; Hanke, Daniela C.; Loessner, Martin J.; Dong, Shengli; Pritchard, David G.; Lee, Jean C.; Becker, Stephen C.; Foster-Frey, Juli; Donovan, David M.

    2015-01-01

    Objectives In the light of increasing drug resistance in Staphylococcus aureus, bacteriophage endolysins [peptidoglycan hydrolases (PGHs)] have been suggested as promising antimicrobial agents. The aim of this study was to determine the antimicrobial activity of nine enzymes representing unique homology groups within a diverse class of staphylococcal PGHs. Methods PGHs were recombinantly expressed, purified and tested for staphylolytic activity in multiple in vitro assays (zymogram, turbidity reduction assay and plate lysis) and against a comprehensive set of strains (S. aureus and CoNS). PGH cut sites in the staphylococcal peptidoglycan were determined by biochemical assays (Park–Johnson and Ghuysen procedures) and MS analysis. The enzymes were tested for their ability to eradicate static S. aureus biofilms and compared for their efficacy against systemic MRSA infection in a mouse model. Results Despite similar modular architectures and unexpectedly conserved cleavage sites in the peptidoglycan (conferred by evolutionarily divergent catalytic domains), the enzymes displayed varying degrees of in vitro lytic activity against numerous staphylococcal strains, including cell surface mutants and drug-resistant strains, and proved effective against static biofilms. In a mouse model of systemic MRSA infection, six PGHs provided 100% protection from death, with animals being free of clinical signs at the end of the experiment. Conclusions Our results corroborate the high potential of PGHs for treatment of S. aureus infections and reveal unique antimicrobial and biochemical properties of the different enzymes, suggesting a high diversity of potential applications despite highly conserved peptidoglycan target sites. PMID:25630640

  7. Structural features of immunologically active polysaccharides from Ganoderma lucidum.

    PubMed

    Bao, Xing-Feng; Wang, Xue-Song; Dong, Qun; Fang, Ji-Nian; Li, Xiao-Yu

    2002-01-01

    Three polysaccharides, two heteroglycans (PL-1 and PL-4) and one glucan (PL-3), were solubilized from the fruit bodies of Ganoderma lucidum and isolated by anion-exchange and gel-filtration chromatography. Their structural features were elucidated by glycosyl residue and glycosyl linkage composition analyses, partial acid hydrolysis, acetolysis, periodate oxidation, 1D and 2D NMR spectroscopy, and ESI-MS experiments. The data obtained indicated that PL-1 had a backbone consisting of 1,4-linked alpha-D-glucopyranosyl residues and 1,6-linked beta-D-galactopyranosyl residues with branches at O-6 of glucose residues and O-2 of galactose residues, composed of terminal glucose, 1,6-linked glucosyl residues and terminal rhamnose. PL-3 was a highly branched glucan composed of 1,3-linked beta-D-glucopyranosyl residues substituted at O-6 with 1,6-linked glucosyl residues. PL-4 was comprised of 1,3-, 1,4-, 1,6-linked beta-D-glucopyranosyl residues and 1,6-linked beta-D-mannopyranosyl residues. These polysaccharides enhanced the proliferation of T- and B-lymphocytes in vitro to varying contents and PL-1 exhibited an immune-stimulating activity in mice. PMID:11809453

  8. Physiological and genomic features of highly alkaliphilic hydrogen-utilizing Betaproteobacteria from a continental serpentinizing site

    PubMed Central

    Suzuki, Shino; Kuenen, J. Gijs; Schipper, Kira; van der Velde, Suzanne; Ishii, Shun’ichi; Wu, Angela; Sorokin, Dimitry Y.; Tenney, Aaron; Meng, XianYing; Morrill, Penny L.; Kamagata, Yoichi; Muyzer, Gerard; Nealson, Kenneth H.

    2014-01-01

    Serpentinization, or the aqueous alteration of ultramafic rocks, results in challenging environments for life in continental sites due to the combination of extremely high pH, low salinity and lack of obvious electron acceptors and carbon sources. Nevertheless, certain Betaproteobacteria have been frequently observed in such environments. Here we describe physiological and genomic features of three related Betaproteobacterial strains isolated from highly alkaline (pH 11.6) serpentinizing springs at The Cedars, California. All three strains are obligate alkaliphiles with an optimum for growth at pH 11 and are capable of autotrophic growth with hydrogen, calcium carbonate and oxygen. The three strains exhibit differences, however, regarding the utilization of organic carbon and electron acceptors. Their global distribution and physiological, genomic and transcriptomic characteristics indicate that the strains are adapted to the alkaline and calcium-rich environments represented by the terrestrial serpentinizing ecosystems. We propose placing these strains in a new genus ‘Serpentinomonas’. PMID:24845058

  9. Identification of S-glutathionylation sites in species-specific proteins by incorporating five sequence-derived features into the general pseudo-amino acid composition.

    PubMed

    Zhao, Xiaowei; Ning, Qiao; Ai, Meiyue; Chai, Haiting; Yang, Guifu

    2016-06-01

    As a selective and reversible protein post-translational modification, S-glutathionylation generates mixed disulfides between glutathione (GSH) and cysteine residues, and plays an important role in regulating protein activity, stability, and redox regulation. To fully understand S-glutathionylation mechanisms, identification of substrates and specific S-Glutathionylated sites is crucial. Experimental identification of S-glutathionylated sites is labor-intensive and time consuming, so establishing an effective computational method is much desirable due to their convenient and fast speed. Therefore, in this study, a new bioinformatics tool named SSGlu (Species-Specific identification of Protein S-glutathionylation Sites) was developed to identify species-specific protein S-glutathionylated sites, utilizing support vector machines that combine multiple sequence-derived features with a two-step feature selection. By 5-fold cross validation, the performance of SSGlu was measured with an AUC of 0.8105 and 0.8041 for Homo sapiens and Mus musculus, respectively. Additionally, SSGlu was compared with the existing methods, and the higher MCC and AUC of SSGlu demonstrated that SSGlu was very promising to predict S-glutathionylated sites. Furthermore, a site-specific analysis showed that S-glutathionylation intimately correlated with the features derived from its surrounding sites. The conclusions derived from this study might help to understand more of the S-glutathionylation mechanism and guide the related experimental validation. For public access, SSGlu is freely accessible at http://59.73.198.144:8080/SSGlu/. PMID:27025952

  10. Quantitative modeling of gene expression using DNA shape features of binding sites.

    PubMed

    Peng, Pei-Chen; Sinha, Saurabh

    2016-07-27

    Prediction of gene expression levels driven by regulatory sequences is pivotal in genomic biology. A major focus in transcriptional regulation is sequence-to-expression modeling, which interprets the enhancer sequence based on transcription factor concentrations and DNA binding specificities and predicts precise gene expression levels in varying cellular contexts. Such models largely rely on the position weight matrix (PWM) model for DNA binding, and the effect of alternative models based on DNA shape remains unexplored. Here, we propose a statistical thermodynamics model of gene expression using DNA shape features of binding sites. We used rigorous methods to evaluate the fits of expression readouts of 37 enhancers regulating spatial gene expression patterns in Drosophila embryo, and show that DNA shape-based models perform arguably better than PWM-based models. We also observed DNA shape captures information complimentary to the PWM, in a way that is useful for expression modeling. Furthermore, we tested if combining shape and PWM-based features provides better predictions than using either binding model alone. Our work demonstrates that the increasingly popular DNA-binding models based on local DNA shape can be useful in sequence-to-expression modeling. It also provides a framework for future studies to predict gene expression better than with PWM models alone. PMID:27257066

  11. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

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

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

  12. Influence of time and length size feature selections for human activity sequences recognition.

    PubMed

    Fang, Hongqing; Chen, Long; Srinivasan, Raghavendiran

    2014-01-01

    In this paper, Viterbi algorithm based on a hidden Markov model is applied to recognize activity sequences from observed sensors events. Alternative features selections of time feature values of sensors events and activity length size feature values are tested, respectively, and then the results of activity sequences recognition performances of Viterbi algorithm are evaluated. The results show that the selection of larger time feature values of sensor events and/or smaller activity length size feature values will generate relatively better results on the activity sequences recognition performances. PMID:24075148

  13. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  14. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

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

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

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

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

    SciTech Connect

    Hartman, F.C.

    1991-01-01

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

  17. A study on the flexibility of enzyme active sites

    PubMed Central

    2011-01-01

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

  18. The effect of recording site on extracted features of motor unit action potential.

    PubMed

    Artuğ, N Tuğrul; Goker, Imran; Bolat, Bülent; Osman, Onur; Kocasoy Orhan, Elif; Baslo, M Baris

    2016-06-01

    Motor unit action potential (MUAP), which consists of individual muscle fiber action potentials (MFAPs), represents the electrical activity of the motor unit. The values of the MUAP features are changed by denervation and reinnervation in neurogenic involvement as well as muscle fiber loss with increased diameter variability in myopathic diseases. The present study is designed to investigate how increased muscle fiber diameter variability affects MUAP parameters in simulated motor units. In order to detect this variation, simulated MUAPs were calculated both at the innervation zone where the MFAPs are more synchronized, and near the tendon, where they show increased temporal dispersion. Reinnervation in neurogenic state increases MUAP amplitude for the recordings at both the innervation zone and near the tendon. However, MUAP duration and the number of peaks significantly increased in a case of myopathy for recordings near the tendon. Furthermore, of the new features, "number of peaks×spike duration" was found as the strongest indicator of MFAP dispersion in myopathy. MUAPs were also recorded from healthy participants in order to investigate the biological counterpart of the simulation data. MUAPs which were recorded near to tendon revealed significantly prolonged duration and decreased amplitude. Although the number of peaks was increased by moving the needle near to tendon, this was not significant. PMID:26817404

  19. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

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

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

    SciTech Connect

    Not Available

    1990-07-01

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

  1. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

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

  2. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

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

  3. Near-surface gas mapping studies of salt geologic features at Weeks Island and other sites

    SciTech Connect

    Molecke, M.A.; Carney, K.R.; Autin, W.J.; Overton, E.B.

    1996-10-01

    Field sampling and rapid gas analysis techniques were used to survey near-surface soil gases for geotechnical diagnostic purposes at the Weeks Island Strategic Petroleum Reserve (SPR) site and other salt dome locations in southern Louisiana. This report presents the complete data, results and interpretations obtained during 1995. Weeks Island 1994 gas survey results are also briefly summarized; this earlier study did not find a definitive correlation between sinkhole No. 1 and soil gases. During 1995, several hundred soil gas samples were obtained and analyzed in the field by gas chromatography, for profiling low concentrations and gas anomalies at ppm to percent levels. The target gases included hydrogen, methane, ethane and ethylene. To supplement the field data, additional gas samples were collected at various site locations for laboratory analysis of target gases at ppb levels. Gases in the near-surface soil originate predominantly from the oil, from petrogenic sources within the salt, or from surface microbial activity. Surveys were conducted across two Weeks Island sinkholes, several mapped anomalous zones in the salt, and over the SPR repository site and its perimeter. Samples were also taken at other south Louisiana salt dome locations for comparative purposes. Notable results from these studies are that elevated levels of hydrogen and methane (1) were positively associated with anomalous gassy or shear zones in the salt dome(s) and (2) are also associated with suspected salt fracture (dilatant) zones over the edges of the SPR repository. Significantly elevated areas of hydrogen, methane, plus some ethane, were found over anomalous shear zones in the salt, particularly in a location over high pressure gas pockets in the salt, identified in the mine prior to SPR operations. Limited stable isotope ratio analyses, SIRA, were also conducted and determined that methane samples were of petrogenic origin, not biogenic.

  4. Habitat Selection in a Rocky Landscape: Experimentally Decoupling the Influence of Retreat Site Attributes from That of Landscape Features

    PubMed Central

    Croak, Benjamin M.; Pike, David A.; Webb, Jonathan K.; Shine, Richard

    2012-01-01

    Organisms selecting retreat sites may evaluate not only the quality of the specific shelter, but also the proximity of that site to resources in the surrounding area. Distinguishing between habitat selection at these two spatial scales is complicated by co-variation among microhabitat factors (i.e., the attributes of individual retreat sites often correlate with their proximity to landscape features). Disentangling this co-variation may facilitate the restoration or conservation of threatened systems. To experimentally examine the role of landscape attributes in determining retreat-site quality for saxicolous ectotherms, we deployed 198 identical artificial rocks in open (sun-exposed) sites on sandstone outcrops in southeastern Australia, and recorded faunal usage of those retreat sites over the next 29 months. Several landscape-scale attributes were associated with occupancy of experimental rocks, but different features were important for different species. For example, endangered broad-headed snakes (Hoplocephalus bungaroides) preferred retreat sites close to cliff edges, flat rock spiders (Hemicloea major) preferred small outcrops, and velvet geckos (Oedura lesueurii) preferred rocks close to the cliff edge with higher-than-average sun exposure. Standardized retreat sites can provide robust experimental data on the effects of landscape-scale attributes on retreat site selection, revealing interspecific divergences among sympatric taxa that use similar habitats. PMID:22701592

  5. Joint Spatial-Spectral Feature Space Clustering for Speech Activity Detection from ECoG Signals

    PubMed Central

    Kanas, Vasileios G.; Mporas, Iosif; Benz, Heather L.; Sgarbas, Kyriakos N.; Bezerianos, Anastasios; Crone, Nathan E.

    2014-01-01

    Brain machine interfaces for speech restoration have been extensively studied for more than two decades. The success of such a system will depend in part on selecting the best brain recording sites and signal features corresponding to speech production. The purpose of this study was to detect speech activity automatically from electrocorticographic signals based on joint spatial-frequency clustering of the ECoG feature space. For this study, the ECoG signals were recorded while a subject performed two different syllable repetition tasks. We found that the optimal frequency resolution to detect speech activity from ECoG signals was 8 Hz, achieving 98.8% accuracy by employing support vector machines (SVM) as a classifier. We also defined the cortical areas that held the most information about the discrimination of speech and non-speech time intervals. Additionally, the results shed light on the distinct cortical areas associated with the two syllable repetition tasks and may contribute to the development of portable ECoG-based communication. PMID:24658248

  6. Active Sites Environmental Monitoring Program FY 1996 annual report

    SciTech Connect

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

    1997-11-01

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

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

    SciTech Connect

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

    1994-05-01

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

  8. Some features of active regions and bursts in millimetric range.

    NASA Astrophysics Data System (ADS)

    Yu, Xingfeng; Yao, Jinxing

    1995-09-01

    The characteristics of active regions and bursts at mm wavelengths, observed with the 13.7 m radio telescope at Quinghai from Nov 16 to Dec 1, 1993, are analyzed. It appears that the active region collapsed and vanished while there occurred a coronal loop with two polarities. GRE bursts at mm wavelength may be interpreted by thermal gyro-resonance radiation and are part of the chromospheric eruption. There is no indication of FFS in 10 ms recordings.

  9. Feature Selection in Classification of Eye Movements Using Electrooculography for Activity Recognition

    PubMed Central

    Mala, S.; Latha, K.

    2014-01-01

    Activity recognition is needed in different requisition, for example, reconnaissance system, patient monitoring, and human-computer interfaces. Feature selection plays an important role in activity recognition, data mining, and machine learning. In selecting subset of features, an efficient evolutionary algorithm Differential Evolution (DE), a very efficient optimizer, is used for finding informative features from eye movements using electrooculography (EOG). Many researchers use EOG signals in human-computer interactions with various computational intelligence methods to analyze eye movements. The proposed system involves analysis of EOG signals using clearness based features, minimum redundancy maximum relevance features, and Differential Evolution based features. This work concentrates more on the feature selection algorithm based on DE in order to improve the classification for faultless activity recognition. PMID:25574185

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2016-06-17

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

  12. Meteorological data for four sites at surface-disruption features in Yucca Flat, Nevada Test Site, Nye County, Nevada, 1985-86

    USGS Publications Warehouse

    Carman, Rita L.

    1994-01-01

    Surface-disruption features, or craters, resulting from underground nuclear testing at the Nevada Test Site may increase the potential for ground-water recharge in an area that would normally produce little, if any, recharge. This report presents selected meteorological data resulting from a study of two surface-disruption features during May 1985 through June 1986. The data were collected at four adjacent sites in Yucca Flat, about 56 kilometers north of Mercury, Nevada. Three sites (one in each of two craters and one at an undisturbed site at the original land surface) were instrumented to collect meteorological data for calculating bare-soil evaporation. These data include (1) long-wave radiation, (2) short-wave radiation, (3) net radiation, (4) air temperae, and (5) soil surface temperature. Meteorological data also were collected at a weather station at an undisturbed site near the study craters. Data collected at this site include (1) air temperature, (2) relative humidity, (3) wind velocity, and (4) wind direction.

  13. Tectonic activity and structural features of active intracontinental normal faults in the Weihe Graben, central China

    NASA Astrophysics Data System (ADS)

    Rao, Gang; Lin, Aiming; Yan, Bing; Jia, Dong; Wu, Xiaojun

    2014-12-01

    This study examines the tectonic activity and structural features of active normal faults in the Weihe Graben, central China. The Weihe Graben is an area with a high level of historic seismicity, and it is one of the intracontinental systems that developed since Tertiary in the extensional environment around the Ordos Block. Analysis of high-resolution remote-sensing imagery data, field observations, and radiocarbon dating results reveal the following: i) active normal faults are mainly developed within a zone < 500 m wide along the southern border of the eastern part of the Weihe Graben; ii) the active faults that have been identified are characterized by stepwise fault scarps dipping into the graben at angles of 40°-71°; iii) there are numerous discontinuous individual fault traces, ranging in length from a few tens of meters to 450 m (generally < 200 m); iv) fault zone structures, topographic features, and fault striations on the main fault planes indicate almost pure normal-slip; and v) late Pleistocene-Holocene terrace risers, loess, and alluvial deposits have been vertically offset by up to ~ 80 m, with a non-uniform dip-slip rate (throw-rates) ranging from ~ 2.1 to 5.7 mm/yr, mostly 2-3 mm/yr. Our results reveal that active normal faults have been developing in the Weihe Graben under an ongoing extensional environment, probably associated with the pre-existing graben and spreading of the continental crust, and this is in contrast with the Ordos Block and neighboring orogenic regions. These results provide new insights into the nature of extensional tectonic deformation in intracontinental graben systems.

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

    SciTech Connect

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

    1986-03-20

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

  15. A small ribozyme with dual-site kinase activity

    PubMed Central

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

    2012-01-01

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

  16. Structural features that specify tyrosine kinase activity deduced from homology modeling of the epidermal growth factor receptor.

    PubMed Central

    Knighton, D R; Cadena, D L; Zheng, J; Ten Eyck, L F; Taylor, S S; Sowadski, J M; Gill, G N

    1993-01-01

    To identify structural features that distinguish protein-tyrosine kinases from protein-serine kinases, a molecular model of the kinase domain of epidermal growth factor receptor was constructed by substituting its amino acid sequence for the amino acid sequence of the catalytic subunit of cAMP-dependent protein kinase in a 2.7-A refined crystallographic model. General folding was conserved as was the configuration of invariant residues at the active site. Two sequence motifs that distinguish the two families correspond to loops that converge at the active site of the enzyme. A conserved arginine in the catalytic loop is proposed to interact with the gamma phosphate of ATP. The second loop provides a binding surface that positions the tyrosine of the substrate. A positively charged surface provides additional sites for substrate recognition. Images Fig. 2 Fig. 3 Fig. 4 PMID:8389462

  17. Evidence for oxygen binding at the active site of particulate methane monooxygenase.

    PubMed

    Culpepper, Megen A; Cutsail, George E; Hoffman, Brian M; Rosenzweig, Amy C

    2012-05-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. The enzyme consists of three subunits, pmoB, pmoA, and pmoC, organized in an α(3)β(3)γ(3) trimer. Studies of intact pMMO and a recombinant soluble fragment of the pmoB subunit (denoted as spmoB) indicate that the active site is located within the soluble region of pmoB at the site of a crystallographically modeled dicopper center. In this work, we have investigated the reactivity of pMMO and spmoB with oxidants. Upon reduction and treatment of spmoB with O(2) or H(2)O(2) or pMMO with H(2)O(2), an absorbance feature at 345 nm is generated. The energy and intensity of this band are similar to those of the μ-η(2):η(2)-peroxo-Cu(II)(2) species formed in several dicopper enzymes and model compounds. The feature is not observed in inactive spmoB variants in which the dicopper center is disrupted, consistent with O(2) binding to the proposed active site. Reaction of the 345 nm species with CH(4) results in the disappearance of the spectroscopic feature, suggesting that this O(2) intermediate is mechanistically relevant. Taken together, these observations provide strong new support for the identity and location of the pMMO active site. PMID:22540911

  18. HIV integration site distributions in resting and activated CD4+ T cells infected in culture

    PubMed Central

    Brady, Troy; Agosto, Luis M.; Malani, Nirav; Berry, Charles C.; O'Doherty, Una; Bushman, Frederic

    2010-01-01

    Objective The goal of this study was to investigate whether the location of HIV integration differs in resting versus activated T cells, a feature that could contribute to the formation of latent viral reservoirs via effects on integration targeting. Design Primary resting or activated CD4+ T cells were infected with purified X4-tropic HIV in the presence and absence of nucleoside triphosphates and genomic locations of integrated provirus determined. Methods We sequenced and analyzed a total of 2661 HIV integration sites using linker-mediated PCR and 454 sequencing. Integration site data sets were then compared to each other and to computationally generated random distributions. Results HIV integration was favored in active transcription units in both cell types, but integration sites from activated cells were found more often in genomic regions that were dense in genes, dense in CpG islands, and enriched in G/C bases. Integration sites from activated cells were also more strongly correlated with histone methylation patterns associated with active genes. Conclusion These data indicate that integration site distributions show modest but significant differences between resting and activated CD4+ T cells, and that integration in resting cells occurs more often in regions that may be suboptimal for proviral gene expression. PMID:19550285

  19. Guaiane sesquiterpenes from Biscogniauxia nummularia featuring potent antigerminative activity.

    PubMed

    Amand, Séverine; Langenfeld, Aude; Blond, Alain; Dupont, Joëlle; Nay, Bastien; Prado, Soizic

    2012-04-27

    Xylaranone, a previously unreported guaiane sesquiterpene along with the known terpenoid xylaranol B and the two mellein derivatives 3,5-dimethyl-8-methoxy-3,4-dihydroisocoumarin and 3,5-dimethyl-8-hydroxy-3,4-dihydroisocoumarin were isolated from Biscogniauxia nummularia. Pogostol was also isolated from this fungus, and in light of our spectroscopic data, its structure was revised and corrected. This fungus, which was isolated as an endophyte from the plum yew Cephalotaxus harringtonia, is also suspected of being a pathogen. Interestingly, we report here the potent antigerminative activity of xylaranone and xylaranol B against seeds of Raphanus sativus at concentrations comparable to glyphosate, a commonly used herbicide. This effect suggests a role for these metabolites in the latent fungal pathogenesis of B. nummularia. PMID:22486738

  20. Structural features important for the biological activity of the potassium channel blocking dendrotoxins.

    PubMed Central

    Hollecker, M.; Marshall, D. L.; Harvey, A. L.

    1993-01-01

    1. Dendrotoxins from mamba snake venoms are small proteins that block neuronal K+ channels. In order to investigate structural features associated with their biological activity, partially folded versions of dendrotoxins I and K from black mamba (Dendroaspis polylepis) were prepared by selectively reducing one or more of their three S-S bonds. 2. The modified toxins were tested for ability to compete with 125I-labelled native toxin I to high affinity binding sites on rat brain synaptosomal membranes and for the ability to increase acetylcholine release in a neuromuscular preparation. 3. Binding affinity increased progressively as the toxins folded to the native conformation and the most biologically active of the modified species were those in which only the disulphide bond between residues 14 and 38 was not formed. These intermediates had native-like conformations as determined by circular dichroism but still had about 5-10 times lower affinity than native toxins. 4. Addition of negatively charged groups to block the free sulthydryls at positions 14 and 38 caused a further, marked loss of activity. 5. The results are consistent with the existence of two important regions in the dendrotoxin molecules. The region containing two of the disulphide bonds (around Cys5-Cys55 and Cys30-Cys51) and much of the secondary structure is essential for the binding affinity of the toxins, while the region around Cys14 and Cys38, equivalent to part of the antiprotease site of the homologous protease inhibitor from bovine pancreas (BPTI), plays an important role in the potency of dendrotoxins. PMID:7694758

  1. Structural features important for the biological activity of the potassium channel blocking dendrotoxins.

    PubMed

    Hollecker, M; Marshall, D L; Harvey, A L

    1993-10-01

    1. Dendrotoxins from mamba snake venoms are small proteins that block neuronal K+ channels. In order to investigate structural features associated with their biological activity, partially folded versions of dendrotoxins I and K from black mamba (Dendroaspis polylepis) were prepared by selectively reducing one or more of their three S-S bonds. 2. The modified toxins were tested for ability to compete with 125I-labelled native toxin I to high affinity binding sites on rat brain synaptosomal membranes and for the ability to increase acetylcholine release in a neuromuscular preparation. 3. Binding affinity increased progressively as the toxins folded to the native conformation and the most biologically active of the modified species were those in which only the disulphide bond between residues 14 and 38 was not formed. These intermediates had native-like conformations as determined by circular dichroism but still had about 5-10 times lower affinity than native toxins. 4. Addition of negatively charged groups to block the free sulthydryls at positions 14 and 38 caused a further, marked loss of activity. 5. The results are consistent with the existence of two important regions in the dendrotoxin molecules. The region containing two of the disulphide bonds (around Cys5-Cys55 and Cys30-Cys51) and much of the secondary structure is essential for the binding affinity of the toxins, while the region around Cys14 and Cys38, equivalent to part of the antiprotease site of the homologous protease inhibitor from bovine pancreas (BPTI), plays an important role in the potency of dendrotoxins. PMID:7694758

  2. Point Cloud Mapping Methods for Documenting Cultural Landscape Features at the Wormsloe State Historic Site, Savannah, Georgia, USA

    NASA Astrophysics Data System (ADS)

    Jordana, T. R.; Goetcheus, C. L.; Madden, M.

    2016-06-01

    Documentation of the three-dimensional (3D) cultural landscape has traditionally been conducted during site visits using conventional photographs, standard ground surveys and manual measurements. In recent years, there have been rapid developments in technologies that produce highly accurate 3D point clouds, including aerial LiDAR, terrestrial laser scanning, and photogrammetric data reduction from unmanned aerial systems (UAS) images and hand held photographs using Structure from Motion (SfM) methods. These 3D point clouds can be precisely scaled and used to conduct measurements of features even after the site visit has ended. As a consequence, it is becoming increasingly possible to collect non-destructive data for a wide variety of cultural site features, including landscapes, buildings, vegetation, artefacts and gardens. As part of a project for the U.S. National Park Service, a variety of data sets have been collected for the Wormsloe State Historic Site, near Savannah, Georgia, USA. In an effort to demonstrate the utility and versatility of these methods at a range of scales, comparisons of the features mapped with different techniques will be discussed with regards to accuracy, data set completeness, cost and ease-of-use.

  3. Quantitative analysis of anthropogenic relief features: automated mapping of charcoal kiln sites from high-resolution ALS data

    NASA Astrophysics Data System (ADS)

    Schneider, Anna; Takla, Melanie; Nicolay, Alexander; Raab, Alexandra; Raab, Thomas

    2014-05-01

    High-resolution digital elevation data from airborne laser scanning (ALS) allow for identification and mapping of so far unknown small-scale relief features that are hidden by forest cover. Especially as a result of historic land use, small anthropogenic landforms can occur, e.g., remains of charcoal kilns on sites that were used for charcoal production or ridge and furrow systems in former farmland areas. Mapping such relief features and analyzing their spatial distribution patterns can help to understand past land-use systems and their effects on landscapes. To efficiently detect and quantify small-scale relief features from high-resolution DEMs for larger areas, (semi-) automated mapping routines are required. In order to describe the number and spatial distribution of historic charcoal kiln sites in the area around Cottbus, Germany, we developed a GIS-based routine for the detection and mapping of kiln remnants from ALS elevation models with a resolution of 1 or 2 meters. The method is based on a template matching algorithm, using a combination of morphometric parameters, and is implemented within ArcGIS. The mapping results could be validated against a comprehensive database of kiln sites and diameters recorded from archaeological excavations in the forefield of the opencast mine Jänschwalde and from manual digitization of kiln remnants from Shaded Relief maps for the Jänschwalder Heide and the Tauersche Forst, north of Cottbus. A considerably high number of charcoal kiln sites could be detected in ALS data, and the diameters of the identified charcoal kilns are remarkable large in the area. For the Jänschwalder Heide, more than 5000 kiln sites in an area of 32 km2 were detected by manual digitization, with 1355 kiln sites that are wider than 12 m. These relatively large kiln sites could be mapped with detection rates that are close to those of manual digitization using the automated mapping routine. Detection quality was improved by the combination of

  4. Dashboard applications to monitor experiment activities at sites

    NASA Astrophysics Data System (ADS)

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

    2010-04-01

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

  5. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

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

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

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

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

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

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

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

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

    PubMed

    Giamarellou, H

    1995-01-01

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

  9. Evidence from molecular dynamics simulations of conformational preorganization in the ribonuclease H active site

    PubMed Central

    Stafford, Kate A.; Palmer III, Arthur G.

    2014-01-01

    Ribonuclease H1 (RNase H) enzymes are well-conserved endonucleases that are present in all domains of life and are particularly important in the life cycle of retroviruses as domains within reverse transcriptase. Despite extensive study, especially of the E. coli homolog, the interaction of the highly negatively charged active site with catalytically required magnesium ions remains poorly understood. In this work, we describe molecular dynamics simulations of the E. coli homolog in complex with magnesium ions, as well as simulations of other homologs in their apo states. Collectively, these results suggest that the active site is highly rigid in the apo state of all homologs studied and is conformationally preorganized to favor the binding of a magnesium ion. Notably, representatives of bacterial, eukaryotic, and retroviral RNases H all exhibit similar active-site rigidity, suggesting that this dynamic feature is only subtly modulated by amino acid sequence and is primarily imposed by the distinctive RNase H protein fold. PMID:25075292

  10. Cosmic ray exposure ages of features and events at the Apollo landing sites

    NASA Technical Reports Server (NTRS)

    Arvidson, R.; Crozaz, G.; Drozd, R. J.; Hohenberg, C. M.; Morgan, C. J.

    1975-01-01

    Cosmic-ray exposure ages of lunar samples have been used to date surface features related to impact cratering and downslope movement of material. Only when multiple samples related to a feature have the same rare-gas exposure age or when a single sample has the same Kr-81 -Kr and track-exposure age can a feature be considered as reliably dated. Based on these criteria, there are only five well-dated lunar features: Cone Crater (Apollo 14), 26 m.y,; North Ray Crater (Apollo 16), 50 m.y.; South Ray Crater (Apollo 16), 2 m.y.; the emplacement of the Station 6 boulders (Apollo 17), 22 m.y.; and the emplacement of the Station 7 boulder (Apollo 17), 28 m.y. Other features are tentatively dated or have limits set on their ages: Bench Crater (Apollo 12), upper limit of 99 m.y.; Baby Ray Crater (Apollo 16), upper limit of 2 m.y.; Shorty Crater (Apollo 17), approximately 30 m.y.; Camelot Crater (Apollo 17) upper limit of 140 m.y.; the emplacement of the Station 2 boulder 1 (Apollo 17), 45 to 55 m.y.; and the slide which generated the light mantle (Apollo 17), lower limit of 50 m.y.

  11. Molecular Imprint of Enzyme Active Site by Camel Nanobodies

    PubMed Central

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

    2012-01-01

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

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

    SciTech Connect

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

    1991-10-01

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

  13. An active-site peptide from pepsin C

    PubMed Central

    Kay, J.; Ryle, A. P.

    1971-01-01

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

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

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

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

  15. Structural and electronic features of a Broensted acid site in H-ZSM-5

    SciTech Connect

    Cook, S.J.; Chakraborty, A.K.; Bell, A.T.; Theodorou, D.N. Univ. of California, Berkeley )

    1993-06-24

    The authors report the results of local density functional theory calculations on a Broensted acid site of the zeolite H-ZSM-5. They have investigated the structural and electronic properties of the site. Comparison is made between their results and existing experimental data. It is shown that structural relaxation around an acid site must be performed to obtain accurate energetics for substitution of aluminum into the zeolite framework. The effects of cluster termination are studied by comparing results obtained for both isolated clusters and clusters embedded in a Madelung field generated by the zeolite framework. The properties of the electron density distribution in the region around an acidic proton indicate that the acidic moiety may be characterized as a rather soft acid. 47 refs., 5 figs., 4 tabs.

  16. Accurate in silico identification of species-specific acetylation sites by integrating protein sequence-derived and functional features

    NASA Astrophysics Data System (ADS)

    Li, Yuan; Wang, Mingjun; Wang, Huilin; Tan, Hao; Zhang, Ziding; Webb, Geoffrey I.; Song, Jiangning

    2014-07-01

    Lysine acetylation is a reversible post-translational modification, playing an important role in cytokine signaling, transcriptional regulation, and apoptosis. To fully understand acetylation mechanisms, identification of substrates and specific acetylation sites is crucial. Experimental identification is often time-consuming and expensive. Alternative bioinformatics methods are cost-effective and can be used in a high-throughput manner to generate relatively precise predictions. Here we develop a method termed as SSPKA for species-specific lysine acetylation prediction, using random forest classifiers that combine sequence-derived and functional features with two-step feature selection. Feature importance analysis indicates functional features, applied for lysine acetylation site prediction for the first time, significantly improve the predictive performance. We apply the SSPKA model to screen the entire human proteome and identify many high-confidence putative substrates that are not previously identified. The results along with the implemented Java tool, serve as useful resources to elucidate the mechanism of lysine acetylation and facilitate hypothesis-driven experimental design and validation.

  17. Function Follows Form: Activation of Shape and Function Features during Object Identification

    ERIC Educational Resources Information Center

    Yee, Eiling; Huffstetler, Stacy; Thompson-Schill, Sharon L.

    2011-01-01

    Most theories of semantic memory characterize knowledge of a given object as comprising a set of semantic features. But how does conceptual activation of these features proceed during object identification? We present the results of a pair of experiments that demonstrate that object recognition is a dynamically unfolding process in which function…

  18. Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

    PubMed

    Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

    2016-08-01

    Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth. PMID:27285815

  19. Feature diagnosticity and task context shape activity in human scene-selective cortex.

    PubMed

    Lowe, Matthew X; Gallivan, Jason P; Ferber, Susanne; Cant, Jonathan S

    2016-01-15

    Scenes are constructed from multiple visual features, yet previous research investigating scene processing has often focused on the contributions of single features in isolation. In the real world, features rarely exist independently of one another and likely converge to inform scene identity in unique ways. Here, we utilize fMRI and pattern classification techniques to examine the interactions between task context (i.e., attend to diagnostic global scene features; texture or layout) and high-level scene attributes (content and spatial boundary) to test the novel hypothesis that scene-selective cortex represents multiple visual features, the importance of which varies according to their diagnostic relevance across scene categories and task demands. Our results show for the first time that scene representations are driven by interactions between multiple visual features and high-level scene attributes. Specifically, univariate analysis of scene-selective cortex revealed that task context and feature diagnosticity shape activity differentially across scene categories. Examination using multivariate decoding methods revealed results consistent with univariate findings, but also evidence for an interaction between high-level scene attributes and diagnostic visual features within scene categories. Critically, these findings suggest visual feature representations are not distributed uniformly across scene categories but are shaped by task context and feature diagnosticity. Thus, we propose that scene-selective cortex constructs a flexible representation of the environment by integrating multiple diagnostically relevant visual features, the nature of which varies according to the particular scene being perceived and the goals of the observer. PMID:26541082

  20. Identifying Key Features of Effective Active Learning: The Effects of Writing and Peer Discussion

    ERIC Educational Resources Information Center

    Linton, Debra L.; Pangle, Wiline M.; Wyatt, Kevin H.; Powell, Karli N.; Sherwood, Rachel E.

    2014-01-01

    We investigated some of the key features of effective active learning by comparing the outcomes of three different methods of implementing active-learning exercises in a majors introductory biology course. Students completed activities in one of three treatments: discussion, writing, and discussion + writing. Treatments were rotated weekly between…

  1. A Consistency-Based Feature Selection Method Allied with Linear SVMs for HIV-1 Protease Cleavage Site Prediction

    PubMed Central

    Öztürk, Orkun; Aksaç, Alper; Elsheikh, Abdallah; Özyer, Tansel; Alhajj, Reda

    2013-01-01

    Background Predicting type-1 Human Immunodeficiency Virus (HIV-1) protease cleavage site in protein molecules and determining its specificity is an important task which has attracted considerable attention in the research community. Achievements in this area are expected to result in effective drug design (especially for HIV-1 protease inhibitors) against this life-threatening virus. However, some drawbacks (like the shortage of the available training data and the high dimensionality of the feature space) turn this task into a difficult classification problem. Thus, various machine learning techniques, and specifically several classification methods have been proposed in order to increase the accuracy of the classification model. In addition, for several classification problems, which are characterized by having few samples and many features, selecting the most relevant features is a major factor for increasing classification accuracy. Results We propose for HIV-1 data a consistency-based feature selection approach in conjunction with recursive feature elimination of support vector machines (SVMs). We used various classifiers for evaluating the results obtained from the feature selection process. We further demonstrated the effectiveness of our proposed method by comparing it with a state-of-the-art feature selection method applied on HIV-1 data, and we evaluated the reported results based on attributes which have been selected from different combinations. Conclusion Applying feature selection on training data before realizing the classification task seems to be a reasonable data-mining process when working with types of data similar to HIV-1. On HIV-1 data, some feature selection or extraction operations in conjunction with different classifiers have been tested and noteworthy outcomes have been reported. These facts motivate for the work presented in this paper. Software availability The software is available at http://ozyer.etu.edu.tr/c-fs-svm.rar. The software

  2. OBSERVED, GIS, AND SELF-REPORTED ENVIRONMENTAL FEATURES AND ADOLESCENT PHYSICAL ACTIVITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Purpose: Examine associations among observed, self-reported, and Geographical Information Systems (GIS) environmental features and physical activity among adolescent males. Design: Cross-sectional study. Setting: Boy Scout troops and neighborhoods in Houston, Texas. Subjects: Two hundred and ten ...

  3. Molecular Features of an Alcohol Binding Site in a Neuronal Potassium Channel†

    PubMed Central

    Shahidullah, Mohammad; Harris, Thanawath; Germann, Markus W.; Covarrubias, Manuel

    2008-01-01

    Aliphatic alcohols (1-alkanols) selectively inhibit the neuronal Shaw2 K+ channel at an internal binding site. This inhibition is conferred by a sequence of 13 residues that constitutes the S4–S5 loop in the pore-forming subunit. Here, we combined functional and structural approaches to gain insights into the molecular basis of this interaction. To infer the forces that are involved, we employed a fast concentration-clamp method (10–90% exchange time = 800 μs) to examine the kinetics of the interaction of three members of the homologous series of 1-alkanols (ethanol, 1-butanol, and 1-hexanol) with Shaw2 K+ channels in Xenopus oocyte inside-out patches. As expected for a second-order mechanism involving a receptor site, only the observed association rate constants were linearly dependent on the 1-alkanol concentration. While the alkyl chain length modestly influenced the dissociation rate constants (decreasing only ∼2-fold between ethanol and 1-hexanol), the second-order association rate constants increased e-fold per carbon atom. Thus, hydrophobic interactions govern the probability of productive collisions at the 1-alkanol binding site, and short-range polar interactions help to stabilize the complex. We also examined the relationship between the energetics of 1-alkanol binding and the structural properties of the S4–S5 loop. Circular dichroism spectroscopy applied to peptides corresponding to the S4–S5 loop of various K+ channels revealed a correlation between the apparent binding affinity of the 1-alkanol binding site and the α-helical propensity of the S4–S5 loop. The data suggest that amphiphilic interactions at the Shaw2 1-alkanol binding site depend on specific structural constraints in the pore-forming subunit of the channel. PMID:14503874

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

    PubMed

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

    1987-11-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  7. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

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

  8. A GIS analysis of suitability for construction aggregate recycling sites using regional transportation network and population density features

    USGS Publications Warehouse

    Robinson, G.R., Jr.; Kapo, K.E.

    2004-01-01

    Aggregate is used in road and building construction to provide bulk, strength, support, and wear resistance. Reclaimed asphalt pavement (RAP) and reclaimed Portland cement concrete (RPCC) are abundant and available sources of recycled aggregate. In this paper, current aggregate production operations in Virginia, Maryland, and the District of Columbia are used to develop spatial association models for the recycled aggregate industry with regional transportation network and population density features. The cost of construction aggregate to the end user is strongly influenced by the cost of transporting processed aggregate from the production site to the construction site. More than 60% of operations recycling aggregate in the mid-Atlantic study area are located within 4.8 km (3 miles) of an interstate highway. Transportation corridors provide both sites of likely road construction where aggregate is used and an efficient means to move both materials and on-site processing equipment back and forth from various work sites to the recycling operations. Urban and developing areas provide a high market demand for aggregate and a ready source of construction debris that may be processed into recycled aggregate. Most aggregate recycling operators in the study area are sited in counties with population densities exceeding 77 people/km2 (200 people/mile 2). No aggregate recycling operations are sited in counties with less than 19 people/km2 (50 people/mile2), reflecting the lack of sufficient long-term sources of construction debris to be used as an aggregate source, as well as the lack of a sufficient market demand for aggregate in most rural areas to locate a recycling operation there or justify the required investment in the equipment to process and produce recycled aggregate. Weights of evidence analyses (WofE), measuring correlation on an area-normalized basis, and weighted logistic regression (WLR), are used to model the distribution of RAP and RPCC operations relative

  9. Energy transfer at the active sites of heme proteins

    SciTech Connect

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

    1995-12-31

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

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

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

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

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

    PubMed

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

    2016-09-01

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

  12. Munitions integrity and corrosion features observed during the HUMMA deep-sea munitions disposal site investigations

    NASA Astrophysics Data System (ADS)

    Silva, Jeff A. K.; Chock, Taylor

    2016-06-01

    An evaluation of the current condition of sea-disposed military munitions observed during the 2009 Hawaii Undersea Military Munitions Assessment Project investigation is presented. The 69 km2 study area is located south of Pearl Harbor, Oahu, Hawaii, and is positioned within a former deep-sea disposal area designated as Hawaii-05 or HI-05 by the United States Department of Defense. HI-05 is known to contain both conventional and chemical munitions that were sea-disposed between 1920 and 1951. Digital images and video reconnaissance logs collected during six remotely operated vehicle and 16 human-occupied vehicle surveys were used to classify the integrity and state of corrosion of the 1842 discarded military munitions (DMM) objects encountered. Of these, 5% (or 90 individual DMM objects) were found to exhibit a mild-moderate degree of corrosion. The majority (66% or 1222 DMM objects) were observed to be significantly corroded, but visually intact on the seafloor. The remaining 29% of DMM encountered were found to be severely corroded and breached, with their contents exposed. Chemical munitions were not identified during the 2009 investigation. In general, identified munitions known to have been constructed with thicker casings were better preserved. Unusual corrosion features were also observed, including what are termed here as 'corrosion skirts' that resembled the flow and cementation of corrosion products at and away from the base of many munitions, and 'corrosion pedestal' features resembling a combination of cemented corrosion products and seafloor sediments that were observed to be supporting munitions above the surface of the seafloor. The origin of these corrosion features could not be determined due to the lack of physical samples collected. However, a microbial-mediated formation hypothesis is presented, based on visual analysis, which can serve as a testable model for future field programs.

  13. Concentric transmon qubit featuring fast tunability and site-selective Z coupling

    NASA Astrophysics Data System (ADS)

    Weides, Martin; Braumueller, Jochen; Sandberg, Martin; Vissers, Michael; Schneider, Andre; Schloer, Steffen; Gruenhaupt, Lukas; Rotzinger, Hannes; Marthaler, Michael; Lukashenko, Alexander; Dieter, Amadeus; Ustinov, Alexey; Pappas, David

    We present a planar qubit design based on a superconducting circuit that we call concentric transmon. While employing a simple fabrication process using Al evaporation and lift-off lithography, we observe qubit lifetimes and coherence times in the order of 10 μs. We systematically characterize loss channels such as incoherent dielectric loss, Purcell decay and radiative losses. The implementation of a gradiometric SQUID loop allows for a fast tuning of the qubit transition frequency and therefore for full tomographic control of the quantum circuit. The presented qubit design features a passive direct Z coupling between neighboring qubits, being a pending quest in the field of quantum simulation.

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

    SciTech Connect

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

    1994-04-01

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

  15. Human activity recognition based on feature selection in smart home using back-propagation algorithm.

    PubMed

    Fang, Hongqing; He, Lei; Si, Hao; Liu, Peng; Xie, Xiaolei

    2014-09-01

    In this paper, Back-propagation(BP) algorithm has been used to train the feed forward neural network for human activity recognition in smart home environments, and inter-class distance method for feature selection of observed motion sensor events is discussed and tested. And then, the human activity recognition performances of neural network using BP algorithm have been evaluated and compared with other probabilistic algorithms: Naïve Bayes(NB) classifier and Hidden Markov Model(HMM). The results show that different feature datasets yield different activity recognition accuracy. The selection of unsuitable feature datasets increases the computational complexity and degrades the activity recognition accuracy. Furthermore, neural network using BP algorithm has relatively better human activity recognition performances than NB classifier and HMM. PMID:25016308

  16. Transform-invariant feature based functional MR image registration and neural activity modelling.

    PubMed

    Gong, Jiaqi; Hao, Qi; Hu, Fei

    2013-01-01

    In this paper, a set of non-rigid image registration and neural activity modelling methods using functional MR Images (fMRI) are proposed based on transform-invariant feature representations. Our work made two contributions. First, we propose to use a transform-invariant feature to improve image registration performance of Iterative Closest Point (ICP) based methods. The proposed feature utilises Gaussian Mixture Models (GMM) to describe the local topological structure of fMRI data. Second, we propose to use a 3-dimensional Scale-Invariant Feature Transform (SIFT) based descriptor to represent neural activities related to drinking behaviour. As a result, neural activities patterns of different subjects drinking water or intaking glucose can be recognised, with strong robustness against various artefacts. PMID:23900434

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

    SciTech Connect

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

    1988-10-18

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

  18. Robust Classification and Segmentation of Planar and Linear Features for Construction Site Progress Monitoring and Structural Dimension Compliance Control

    NASA Astrophysics Data System (ADS)

    Maalek, R.; Lichti, D. D.; Ruwanpura, J.

    2015-08-01

    The application of terrestrial laser scanners (TLSs) on construction sites for automating construction progress monitoring and controlling structural dimension compliance is growing markedly. However, current research in construction management relies on the planned building information model (BIM) to assign the accumulated point clouds to their corresponding structural elements, which may not be reliable in cases where the dimensions of the as-built structure differ from those of the planned model and/or the planned model is not available with sufficient detail. In addition outliers exist in construction site datasets due to data artefacts caused by moving objects, occlusions and dust. In order to overcome the aforementioned limitations, a novel method for robust classification and segmentation of planar and linear features is proposed to reduce the effects of outliers present in the LiDAR data collected from construction sites. First, coplanar and collinear points are classified through a robust principal components analysis procedure. The classified points are then grouped using a robust clustering method. A method is also proposed to robustly extract the points belonging to the flat-slab floors and/or ceilings without performing the aforementioned stages in order to preserve computational efficiency. The applicability of the proposed method is investigated in two scenarios, namely, a laboratory with 30 million points and an actual construction site with over 150 million points. The results obtained by the two experiments validate the suitability of the proposed method for robust segmentation of planar and linear features in contaminated datasets, such as those collected from construction sites.

  19. SRAMP: prediction of mammalian N6-methyladenosine (m6A) sites based on sequence-derived features.

    PubMed

    Zhou, Yuan; Zeng, Pan; Li, Yan-Hui; Zhang, Ziding; Cui, Qinghua

    2016-06-01

    N(6)-methyladenosine (m(6)A) is a prevalent RNA methylation modification involved in the regulation of degradation, subcellular localization, splicing and local conformation changes of RNA transcripts. High-throughput experiments have demonstrated that only a small fraction of the m(6)A consensus motifs in mammalian transcriptomes are modified. Therefore, accurate identification of RNA m(6)A sites becomes emergently important. For the above purpose, here a computational predictor of mammalian m(6)A site named SRAMP is established. To depict the sequence context around m(6)A sites, SRAMP combines three random forest classifiers that exploit the positional nucleotide sequence pattern, the K-nearest neighbor information and the position-independent nucleotide pair spectrum features, respectively. SRAMP uses either genomic sequences or cDNA sequences as its input. With either kind of input sequence, SRAMP achieves competitive performance in both cross-validation tests and rigorous independent benchmarking tests. Analyses of the informative features and overrepresented rules extracted from the random forest classifiers demonstrate that nucleotide usage preferences at the distal positions, in addition to those at the proximal positions, contribute to the classification. As a public prediction server, SRAMP is freely available at http://www.cuilab.cn/sramp/. PMID:26896799

  20. SRAMP: prediction of mammalian N6-methyladenosine (m6A) sites based on sequence-derived features

    PubMed Central

    Zhou, Yuan; Zeng, Pan; Li, Yan-Hui; Zhang, Ziding; Cui, Qinghua

    2016-01-01

    N6-methyladenosine (m6A) is a prevalent RNA methylation modification involved in the regulation of degradation, subcellular localization, splicing and local conformation changes of RNA transcripts. High-throughput experiments have demonstrated that only a small fraction of the m6A consensus motifs in mammalian transcriptomes are modified. Therefore, accurate identification of RNA m6A sites becomes emergently important. For the above purpose, here a computational predictor of mammalian m6A site named SRAMP is established. To depict the sequence context around m6A sites, SRAMP combines three random forest classifiers that exploit the positional nucleotide sequence pattern, the K-nearest neighbor information and the position-independent nucleotide pair spectrum features, respectively. SRAMP uses either genomic sequences or cDNA sequences as its input. With either kind of input sequence, SRAMP achieves competitive performance in both cross-validation tests and rigorous independent benchmarking tests. Analyses of the informative features and overrepresented rules extracted from the random forest classifiers demonstrate that nucleotide usage preferences at the distal positions, in addition to those at the proximal positions, contribute to the classification. As a public prediction server, SRAMP is freely available at http://www.cuilab.cn/sramp/. PMID:26896799

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

    PubMed

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

    2007-06-26

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

  2. Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins.

    PubMed

    Domenighini, M; Magagnoli, C; Pizza, M; Rappuoli, R

    1994-10-01

    Computer analysis of the three-dimensional structure of ADP-ribosylating toxins showed that in all toxins the NAD-binding site is located in a cavity. This cavity consists of 18 contiguous amino acids that form an alpha-helix bent over a beta-strand. The tertiary folding of this structure is strictly conserved despite the differences in the amino acid sequence. Catalysis is supported by two spatially conserved amino acids, each flanking the NAD-binding site. These are: a glutamic acid that is conserved in all toxins, and a nucleophilic residue, which is a histidine in the diphtheria toxin and Pseudomonas exotoxin A, and an arginine in the cholera toxin, the Escherichia coli heat-labile enterotoxins, the pertussis toxin and the mosquitocidal toxin of Bacillus sphaericus. The latter group of toxins presents an additional histidine that appears important for catalysis. This structure suggests a general mechanism of ADP-ribosylation evolved to work on different target proteins. PMID:7830559

  3. Emotion Recognition with Eigen Features of Frequency Band Activities Embedded in Induced Brain Oscillations Mediated by Affective Pictures.

    PubMed

    Aydin, Serap; Demirtaş, Serdar; Ateş, Kahraman; Tunga, M Alper

    2016-05-01

    In this study, singular spectrum analysis (SSA) has been used for the first time in order to extract emotional features from well-defined electroencephalography (EEG) frequency band activities (BAs) so-called delta (0.5-4 Hz), theta (4-8 Hz), alpha (8-16 Hz), beta (16-32 Hz), gamma (32-64 Hz). These five BAs were estimated by applying sixth-level multi-resolution wavelet decomposition (MRWD) with Daubechies wavelets (db-8) to single channel nonaveraged emotional EEG oscillations of 6 s for each scalp location over 16 recording sites (Fp1, Fp2, F3, F4, F7, F8, C3, C4, P3, P4, T3, T4, T5, T6, O1, O2). Every trial was mediated by different emotional stimuli which were selected from international affective picture system (IAPS) to induce emotional states such as pleasant (P), neutral (N), and unpleasant (UP). Largest principal components (PCs) of BAs were considered as emotional features and data mining approaches were used for the first time in order to classify both three different (P, N, UP) and two contrasting (P and UP) emotional states for 30 healthy controls. Emotional features extracted from gamma BAs (GBAs) for 16 recording sites provided the high classification accuracies of 87.1% and 100% for classification of three emotional states and two contrasting emotional states, respectively. In conclusion, we found the followings: (1) Eigenspectra of high frequency BAs in EEG are highly sensitive to emotional hemispheric activations, (2) emotional states are mostly mediated by GBA, (3) pleasant pictures induce the higher cortical activation in contrast to unpleasant pictures, (4) contrasting emotions induce opposite cortical activations, (5) cognitive activities are necessary for an emotion to occur. PMID:26971786

  4. DNA binding induces active site conformational change in the human TREX2 3'-exonuclease.

    PubMed

    de Silva, Udesh; Perrino, Fred W; Hollis, Thomas

    2009-04-01

    The TREX enzymes process DNA as the major 3'-->5' exonuclease activity in mammalian cells. TREX2 and TREX1 are members of the DnaQ family of exonucleases and utilize a two metal ion catalytic mechanism of hydrolysis. The structure of the dimeric TREX2 enzyme in complex with single-stranded DNA has revealed binding properties that are distinct from the TREX1 protein. The TREX2 protein undergoes a conformational change in the active site upon DNA binding including ordering of active site residues and a shift of an active site helix. Surprisingly, even when a single monomer binds DNA, both monomers in the dimer undergo the structural rearrangement. From this we have proposed a model for DNA binding and 3' hydrolysis for the TREX2 dimer. The structure also shows how TREX proteins potentially interact with double-stranded DNA and suggest features that might be involved in strand denaturation to provide a single-stranded substrate for the active site. PMID:19321497

  5. RELATIONSHIPS BETWEEN GIS ENVIRONMENTAL FEATURES AND ADOLESCENT MALE PHYSICAL ACTIVITY: GIS CODING DIFFERENCES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: It is not clear if relationships between GIS obtained environmental features and physical activity differ according to the method used to code GIS data. Methods: Physical activity levels of 210 Boy Scouts were measured by accelerometer. Numbers of parks, trails, gymnasia, bus stops, groc...

  6. Co-Conserved MAPK Features Couple D-Domain Docking Groove to Distal Allosteric Sites via the C-Terminal Flanking Tail

    PubMed Central

    Nguyen, Tuan; Ruan, Zheng; Oruganty, Krishnadev; Kannan, Natarajan

    2015-01-01

    Mitogen activated protein kinases (MAPKs) form a closely related family of kinases that control critical pathways associated with cell growth and survival. Although MAPKs have been extensively characterized at the biochemical, cellular, and structural level, an integrated evolutionary understanding of how MAPKs differ from other closely related protein kinases is currently lacking. Here, we perform statistical sequence comparisons of MAPKs and related protein kinases to identify sequence and structural features associated with MAPK functional divergence. We show, for the first time, that virtually all MAPK-distinguishing sequence features, including an unappreciated short insert segment in the β4-β5 loop, physically couple distal functional sites in the kinase domain to the D-domain peptide docking groove via the C-terminal flanking tail (C-tail). The coupling mediated by MAPK-specific residues confers an allosteric regulatory mechanism unique to MAPKs. In particular, the regulatory αC-helix conformation is controlled by a MAPK-conserved salt bridge interaction between an arginine in the αC-helix and an acidic residue in the C-tail. The salt-bridge interaction is modulated in unique ways in individual sub-families to achieve regulatory specificity. Our study is consistent with a model in which the C-tail co-evolved with the D-domain docking site to allosterically control MAPK activity. Our study provides testable mechanistic hypotheses for biochemical characterization of MAPK-conserved residues and new avenues for the design of allosteric MAPK inhibitors. PMID:25799139

  7. Preliminary Safety Analysis of the Gorleben Site: Safety Concept and Application to Scenario Development Based on a Site-Specific Features, Events and Processes (FEP) Database - 13304

    SciTech Connect

    Moenig, Joerg; Beuth, Thomas; Wolf, Jens; Lommerzheim, Andre; Mrugalla, Sabine

    2013-07-01

    Based upon the German safety criteria, released in 2010 by the Federal Ministry of the Environment (BMU), a safety concept and a safety assessment concept for the disposal of heat-generating high-level waste have both been developed in the framework of the preliminary safety case for the Gorleben site (Project VSG). The main objective of the disposal is to contain the radioactive waste inside a defined rock zone, which is called containment-providing rock zone. The radionuclides shall remain essentially at the emplacement site, and at the most, a small defined quantity of material shall be able to leave this rock zone. This shall be accomplished by the geological barrier and a technical barrier system, which is required to seal the inevitable penetration of the geological barrier by the construction of the mine. The safe containment has to be demonstrated for probable and less probable evolutions of the site, while evolutions with very low probability (less than 1 % over the demonstration period of 1 million years) need not to be considered. Owing to the uncertainty in predicting the real evolution of the site, plausible scenarios have been derived in a systematic manner. Therefore, a comprehensive site-specific features, events and processes (FEP) data base for the Gorleben site has been developed. The safety concept was directly taken into account, e.g. by identification of FEP with direct influence on the barriers that provide the containment. No effort was spared to identify the interactions of the FEP, their probabilities of occurrence, and their characteristics (values). The information stored in the data base provided the basis for the development of scenarios. The scenario development methodology is based on FEP related to an impairment of the functionality of a subset of barriers, called initial barriers. By taking these FEP into account in their probable characteristics the reference scenario is derived. Thus, the reference scenario describes a

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

    USGS Publications Warehouse

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

    2000-01-01

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

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

    PubMed Central

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

    2000-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  11. Site History and Edaphic Features Override the Influence of Plant Species on Microbial Communities in Restored Tidal Freshwater Wetlands

    PubMed Central

    Prasse, Christine E.; Baldwin, Andrew H.

    2015-01-01

    Restored wetland soils differ significantly in physical and chemical properties from their natural counterparts even when plant community compositions are similar, but effects of restoration on microbial community composition and function are not well understood. Here, we investigate plant-microbe relationships in restored and natural tidal freshwater wetlands from two subestuaries of the Chesapeake Bay. Soil samples were collected from the root zone of Typha latifolia, Phragmites australis, Peltandra virginica, and Lythrum salicaria. Soil microbial composition was assessed using 454 pyrosequencing, and genes representing bacteria, archaea, denitrification, methanogenesis, and methane oxidation were quantified. Our analysis revealed variation in some functional gene copy numbers between plant species within sites, but intersite comparisons did not reveal consistent plant-microbe trends. We observed more microbial variations between plant species in natural wetlands, where plants have been established for a long period of time. In the largest natural wetland site, sequences putatively matching methanogens accounted for ∼17% of all sequences, and the same wetland had the highest numbers of genes coding for methane coenzyme A reductase (mcrA). Sequences putatively matching aerobic methanotrophic bacteria and anaerobic methane-oxidizing archaea (ANME) were detected in all sites, suggesting that both aerobic and anaerobic methane oxidation are possible in these systems. Our data suggest that site history and edaphic features override the influence of plant species on microbial communities in restored wetlands. PMID:25769832

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

    SciTech Connect

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

    1994-08-01

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

  13. Identification of active sites in gold-catalyzed hydrogenation of acrolein.

    PubMed

    Mohr, Christian; Hofmeister, Herbert; Radnik, Jörg; Claus, Peter

    2003-02-19

    The active sites of supported gold catalysts, favoring the adsorption of C=O groups of acrolein and subsequent reaction to allyl alcohol, have been identified as edges of gold nanoparticles. After our recent finding that this reaction preferentially occurs on single crystalline particles rather than multiply twinned ones, this paper reports on a new approach to distinguish different features of the gold particle morphology. Elucidation of the active site issue cannot be simply done by varying the size of gold particles, since the effects of faceting and multiply twinned particles may interfere. Therefore, modification of the gold particle surface by indium has been used to vary the active site characteristics of a suitable catalyst, and a selective decoration of gold particle faces has been observed, leaving edges free. This is in contradiction to theoretical predictions, suggesting a preferred occupation of the low-coordinated edges of the gold particles. On the bimetallic catalyst, the desired allyl alcohol is the main product (selectivity 63%; temperature 593 K, total pressure p(total) = 2 MPa). From the experimentally proven correlation between surface structure and catalytic behavior, the edges of single crystalline gold particles have been identified as active sites for the preferred C=O hydrogenation. PMID:12580618

  14. Feature activation during word recognition: action, visual, and associative-semantic priming effects

    PubMed Central

    Lam, Kevin J. Y.; Dijkstra, Ton; Rueschemeyer, Shirley-Ann

    2015-01-01

    Embodied theories of language postulate that language meaning is stored in modality-specific brain areas generally involved in perception and action in the real world. However, the temporal dynamics of the interaction between modality-specific information and lexical-semantic processing remain unclear. We investigated the relative timing at which two types of modality-specific information (action-based and visual-form information) contribute to lexical-semantic comprehension. To this end, we applied a behavioral priming paradigm in which prime and target words were related with respect to (1) action features, (2) visual features, or (3) semantically associative information. Using a Go/No-Go lexical decision task, priming effects were measured across four different inter-stimulus intervals (ISI = 100, 250, 400, and 1000 ms) to determine the relative time course of the different features. Notably, action priming effects were found in ISIs of 100, 250, and 1000 ms whereas a visual priming effect was seen only in the ISI of 1000 ms. Importantly, our data suggest that features follow different time courses of activation during word recognition. In this regard, feature activation is dynamic, measurable in specific time windows but not in others. Thus the current study (1) demonstrates how multiple ISIs can be used within an experiment to help chart the time course of feature activation and (2) provides new evidence for embodied theories of language. PMID:26074836

  15. Potential sites of CFTR activation by tyrosine kinases.

    PubMed

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

    2016-05-01

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

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

    SciTech Connect

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

    2014-08-14

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

  17. In vitro genetic analysis of the structural features of the pre-tRNA required for determination of the 3' splice site in the intron excision reaction.

    PubMed Central

    Bufardeci, E; Fabbri, S; Baldi, M I; Mattoccia, E; Tocchini-Valentini, G P

    1993-01-01

    During processing of intron-containing pre-tRNAs, the Xenopus laevis splicing endonuclease binds the precursor and cleaves it at both the 5' and 3' splice sites. In vitro selection was used to determine structural features characteristic of precursor tRNA molecules that are active in this reaction. We performed two types of selection, one for molecules that are not cut, the other for molecules that are cut at only one site. The results shed light on various aspects of the intron excision reaction, including the importance of the three-dimensional structure of the mature domain for recognition and binding of the enzyme, the active role played by the single-stranded region of the intron, and the importance of the cardinal positions which, although not necessarily occupied by the same base in all precursors, nevertheless play a fundamental role in the splicing reaction. A precursor can be cut at the 3' site if a base in the single-stranded loop of the intron is allowed to pair (A-I pair) with the base of the 5' exon situated at the position immediately following the anticodon stem [first cardinal position (CP1)]. The nature of the bases involved in the A-I pair is important, as is the position of the base in the single-stranded loop of the intron. We discuss the role of the cardinal positions in the reaction. Images PMID:8223479

  18. A concept for extraction of habitat features from laser scanning and hypersprectral imaging for evaluation of Natura 2000 sites - the ChangeHabitats2 project approach

    NASA Astrophysics Data System (ADS)

    Székely, B.; Kania, A.; Pfeifer, N.; Heilmeier, H.; Tamás, J.; Szöllősi, N.; Mücke, W.

    2012-04-01

    The goal of the ChangeHabitats2 project is the development of cost- and time-efficient habitat assessment strategies by employing effective field work techniques supported by modern airborne remote sensing methods, i.e. hyperspectral imagery and laser scanning (LiDAR). An essential task of the project is the design of a novel field work technique that on the one hand fulfills the reporting requirements of the Flora-Fauna-Habitat (FFH-) directive and on the other hand serves as a reference for the aerial data analysis. Correlations between parameters derived from remotely sensed data and terrestrial field measurements shall be exploited in order to create half- or fully-automated methods for the extraction of relevant Natura2000 habitat parameters. As a result of these efforts a comprehensive conceptual model has been developed for extraction and integration of Natura 2000 relevant geospatial data. This scheme is an attempt to integrate various activities within ChangeHabitats2 project defining pathways of development, as well as encompassing existing data processing chains, theoretical approaches and field work. The conceptual model includes definition of processing levels (similar to those existing in remote sensing), whereas these levels cover the range from the raw data to the extracted habitat feature. For instance, the amount of dead wood (standing or lying on the surface) is an important evaluation criterion for the habitat. The tree trunks lying on the ground surface typically can be extracted from the LiDAR point cloud, and the amount of wood can be estimated accordingly. The final result will be considered as a habitat feature derived from laser scanning data. Furthermore, we are also interested not only in the determination of the specific habitat feature, but also in the detection of its variations (especially in deterioration). In this approach the variation of this important habitat feature is considered to be a differential habitat feature, that can

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

    SciTech Connect

    1981-02-27

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

  20. Using Feature Films To Promote Active Learning in the College Classroom.

    ERIC Educational Resources Information Center

    Gregg, Virginia R.; And Others

    Using feature films to teach undergraduate psychology courses can promote active learning for several reasons. Films can reach students with a variety of learning styles, including those with a visual approach to learning. Also, students seem to enjoy commercial films and their use can help decrease levels of monotony from daily lectures. Feature…

  1. From Monty Python to Total Recall: A Feature Film Activity for the Cognitive Psychology Course.

    ERIC Educational Resources Information Center

    Conner, David B.

    1996-01-01

    Describes a college psychology course activity designed to help students define the parameters of cognitive psychology. Students selected a feature film and a journal article that represented some aspect of cognitive psychology. They then wrote a paper discussing the theoretical and empirical connections between the sources and the topic. (MJP)

  2. Recognition of forearm muscle activity by continuous classification of multi-site mechanomyogram signals.

    PubMed

    Alves, Natasha; Chau, Tom

    2010-01-01

    Recent studies on identifying multiple activation states from mechanomyogram (MMG) signals for the purpose of controlling switching interfaces have employed pattern recognition methods where MMG signal features from multiple muscle sites are extracted and classified. The purpose of this study is to determine if MMG signal features retain enough discriminatory information to allow reliable continuous classification, and to determine if there is a decline in classification accuracy over short time periods. MMG signals were recorded from two accelerometers attached to the flexor carpi radialis and extensor carpi radialis muscles of 12 able-bodied participants as participants performed three classes of forearm muscle activity. The data were collected over five recording sessions, with a ten-minute interval between each session. The data were spliced into 256 ms epochs, and a comprehensive set of signal features was extracted. A pattern classifier, trained with continuously acquired signal features from the first recording session, was tested with signals recorded from the other sessions. The average classification accuracy over the five sessions was 89 ± 2%. There was no obvious declining trend in classification accuracy with time. These results show that MMG signals recorded at the forearm retain enough discriminatory information to allow continuous recognition of hand motion across multiple (>90) repetitions, and the MMG-classifier does not show short-term degradation. These results indicate the potential of MMG as a multifunction control signal for muscle-machine interfaces. PMID:21097038

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

    SciTech Connect

    Carlton, W.H.; Denham, M.

    1996-07-01

    This document assesses the impact of radioactive activation products released from SRS facilities since the first reactor became operational late in 1953. The isotopes reported here are those whose release resulted in the highest dose to people living near SRS: {sup 32}P, {sup 51}Cr, {sup 60}C, and {sup 65}Zn. Release pathways, emission control features, and annual releases to the aqueous and atmospheric environments are discussed. No single incident has resulted in a major acute release of activation products to the environment. The releases were the result of normal operations of the reactors and separations facilities. Releases declined over the years as better controls were established and production was reduced. The overall radiological impact of SRS activation product atmospheric releases from 1954 through 1994 on the offsite maximally exposed individual can be characterized by a total dose of 0.76 mrem. During the same period, such an individual received a total dose of 14,400 mrem from non-SRS sources of ionizing radiation present in the environment. SRS activation product aqueous releases between 1954 and 1994 resulted in a total dose of 54 mrem to the offsite maximally exposed individual. The impact of SRS activation product releases on offsite populations also has been evaluated.

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

    PubMed Central

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

    2015-01-01

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

  5. Feature Statistics Modulate the Activation of Meaning During Spoken Word Processing.

    PubMed

    Devereux, Barry J; Taylor, Kirsten I; Randall, Billi; Geertzen, Jeroen; Tyler, Lorraine K

    2016-03-01

    Understanding spoken words involves a rapid mapping from speech to conceptual representations. One distributed feature-based conceptual account assumes that the statistical characteristics of concepts' features--the number of concepts they occur in (distinctiveness/sharedness) and likelihood of co-occurrence (correlational strength)--determine conceptual activation. To test these claims, we investigated the role of distinctiveness/sharedness and correlational strength in speech-to-meaning mapping, using a lexical decision task and computational simulations. Responses were faster for concepts with higher sharedness, suggesting that shared features are facilitatory in tasks like lexical decision that require access to them. Correlational strength facilitated responses for slower participants, suggesting a time-sensitive co-occurrence-driven settling mechanism. The computational simulation showed similar effects, with early effects of shared features and later effects of correlational strength. These results support a general-to-specific account of conceptual processing, whereby early activation of shared features is followed by the gradual emergence of a specific target representation. PMID:26043761

  6. Using Activity-Related Behavioural Features towards More Effective Automatic Stress Detection

    PubMed Central

    Giakoumis, Dimitris; Drosou, Anastasios; Cipresso, Pietro; Tzovaras, Dimitrios; Hassapis, George; Gaggioli, Andrea; Riva, Giuseppe

    2012-01-01

    This paper introduces activity-related behavioural features that can be automatically extracted from a computer system, with the aim to increase the effectiveness of automatic stress detection. The proposed features are based on processing of appropriate video and accelerometer recordings taken from the monitored subjects. For the purposes of the present study, an experiment was conducted that utilized a stress-induction protocol based on the stroop colour word test. Video, accelerometer and biosignal (Electrocardiogram and Galvanic Skin Response) recordings were collected from nineteen participants. Then, an explorative study was conducted by following a methodology mainly based on spatiotemporal descriptors (Motion History Images) that are extracted from video sequences. A large set of activity-related behavioural features, potentially useful for automatic stress detection, were proposed and examined. Experimental evaluation showed that several of these behavioural features significantly correlate to self-reported stress. Moreover, it was found that the use of the proposed features can significantly enhance the performance of typical automatic stress detection systems, commonly based on biosignal processing. PMID:23028461

  7. Orientations and other features of the Neolithic 'giants' churches' of Finland from on-site and lidar observations

    NASA Astrophysics Data System (ADS)

    Ridderstad, M. P.

    2015-07-01

    The orientations and placement of 52 Neolithic stone enclosures in Finland known as 'Giants' Churches' were analysed. In addition, other characteristic features, such as cairns and standing stones in or near the Giants' Churches, were investigated. The axis and gate orientations of the structures were measured using both on-site and airborne laser scanning (lidar) observations. The results showed lidar observations to be useful in archaeoastronomical analysis as a complementary tool to be used with on-site measurements and observations. The Giants' Churches were found to be orientations towards certain solar and lunar events that could have acted as 'seasonal pointers'. The orientations of the gates of the GCs were found to replicate the axis orientations to a large degree. The majority (over 90%) of the GCs were positioned on the eastern or southeastern side of the ridge they were built on, indicating the interest of the builders in the eastern horizon and possibly the rising of celestial bodies. The orientations of large (>35-m long) Giants' Churches and small (≤35-m long) ones were compared. The observed differences in the orientations of these two groups suggested that the structures traditionally known as Giants' Churches may be a heterogeneous group consisting of at least two types of structures represented in this study by the two selected size groups. Many large GCs were found to have been oriented towards the solstices, while the smaller ones did not show this feature. It is possible that the smaller Giants' Churches were oriented towards the Moon, while the larger ones were associated to solar events. The smaller Giants' Churches could be the remains of large houses or otherwise belong to a different tradition of construction.

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

    PubMed

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

  11. Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

    PubMed

    Prado, R A; Barbosa, J A; Ohmiya, Y; Viviani, V R

    2011-07-01

    The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases. PMID:21505686

  12. Redd site selection and spawning habitat use by fall chinook salmon: The importance of geomorphic features in large rivers

    SciTech Connect

    Geist, D.R. |; Dauble, D.D.

    1998-09-01

    Knowledge of the three-dimensional connectivity between rivers and groundwater within the hyporheic zone can be used to improve the definition of fall chinook salmon (Oncorhynchus tshawytscha) spawning habitat. Information exists on the microhabitat characteristics that define suitable salmon spawning habitat. However, traditional spawning habitat models that use these characteristics to predict available spawning habitat are restricted because they can not account for the heterogeneous nature of rivers. The authors present a conceptual spawning habitat model for fall chinook salmon that describes how geomorphic features of river channels create hydraulic processes, including hyporheic flows, that influence where salmon spawn in unconstrained reaches of large mainstem alluvial rivers. Two case studies based on empirical data from fall chinook salmon spawning areas in the Hanford Reach of the Columbia River are presented to illustrate important aspects of the conceptual model. The authors suggest that traditional habitat models and the conceptual model be combined to predict the limits of suitable fall chinook salmon spawning habitat. This approach can incorporate quantitative measures of river channel morphology, including general descriptors of geomorphic features at different spatial scales, in order to understand the processes influencing redd site selection and spawning habitat use. This information is needed in order to protect existing salmon spawning habitat in large rivers, as well as to recover habitat already lost.

  13. The copper active site of CBM33 polysaccharide oxygenases.

    PubMed

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

    2013-04-24

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

  14. The Copper Active Site of CBM33 Polysaccharide Oxygenases

    PubMed Central

    2013-01-01

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

  15. FUNCTION FOLLOWS FORM: ACTIVATION OF SHAPE & FUNCTION FEATURES DURING OBJECT IDENTIFICATION

    PubMed Central

    Yee, Eiling; Huffstetler, Stacy; Thompson-Schill, Sharon L.

    2011-01-01

    Most theories of semantic memory characterize knowledge of a given object as comprising a set of semantic features. But how does conceptual activation of these features proceed during object identification? We present the results of a pair of experiments that demonstrate that object recognition is a dynamically unfolding process in which function follows form. We used eye movements to explore whether activating one object’s concept leads to the activation of others that share perceptual (shape) or abstract (function) features. Participants viewed four-picture displays and clicked on the picture corresponding to a heard word. In critical trials, the conceptual representation of one of the objects in the display was similar in shape or function (i.e., its purpose) to the heard word. Importantly, this similarity was not apparent in the visual depictions (e.g., for the target “frisbee,” the shape-related object was a triangular slice of pizza – a shape that a frisbee cannot take); preferential fixations on the related object were therefore attributable to overlap of the conceptual representations on the relevant features. We observed relatedness effects for both shape and function, but shape effects occurred earlier than function effects. We discuss the implications of these findings for current accounts of the representation of semantic memory. PMID:21417543

  16. Multifractal features of magnetospheric dynamics and their dependence on solar activity

    NASA Astrophysics Data System (ADS)

    Gopinath, Sumesh

    2016-09-01

    In the present study, novel wavelet leaders (WL) based multifractal analysis has been used to get a better knowledge of the self-organization phenomena inherent in complex magnetospheric dynamics during disturbance and quiescent periods, focusing mainly on the intermittent features of auroral electrojet (AE) index. The results derived from the analysis certainly exhibit the phase transition property of magnetosphere system with respect to variabilities in the driving conditions. By using the novel WL method, solar activity dependence/independence of intermittency of magnetospheric proxies such as AE, SYM-H and Dst indices have been compared. The results indicate that the multifractality of AE index does not follow the solar activity cycle while intermittent features of SYM-H and Dst indices show high degree of solar activity dependence. This shows that along with the external solar wind perturbations, certain complex phenomena of internal origin also significantly modulate the dynamics of geomagnetic fluctuations in the auroral region.

  17. North-south asymmetry of different solar activity features during solar cycle 23

    NASA Astrophysics Data System (ADS)

    Bankoti, Neeraj Singh; Joshi, Navin Chandra; Pande, Seema; Pande, Bimal; Pandey, Kavita

    2010-08-01

    A study on north-south (N-S) asymmetry of different solar activity features (DSAF) such as solar proton events, solar active prominences [total, low (⩽40°) and high (⩾50°) latitudes], H α flare indices, soft X-ray flares, monthly mean sunspot areas and monthly mean sunspot numbers carried out from May 1996 to October 2008. Study shows a southern dominance of DSAF during this period. During the rising phase of the cycle 23 the number of DSAF approximately equals on both, the northern and the southern hemispheres. But these activities tend to shift from northern to southern hemisphere during the period 1998-1999. The statistical significance of the asymmetry time series using a χ2-test of goodness of fit indicates that in most of the cases the asymmetry is highly significant, meaning thereby that the asymmetry is a real feature in the N-S distribution of DSAF.

  18. Optimal feature point selection and automatic initialization in active shape model search.

    PubMed

    Lekadir, Karim; Yang, Guang-Zhong

    2008-01-01

    This paper presents a novel approach for robust and fully automatic segmentation with active shape model search. The proposed method incorporates global geometric constraints during feature point search by using interlandmark conditional probabilities. The A* graph search algorithm is adapted to identify in the image the optimal set of valid feature points. The technique is extended to enable reliable and fast automatic initialization of the ASM search. Validation with 2-D and 3-D MR segmentation of the left ventricular epicardial border demonstrates significant improvement in robustness and overall accuracy, while eliminating the need for manual initialization. PMID:18979776

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

    SciTech Connect

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

    2010-08-13

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

  20. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    SciTech Connect

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

    2008-04-02

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

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

    SciTech Connect

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

    1986-05-01

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

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

    PubMed

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

    2016-04-22

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

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

    SciTech Connect

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

    1986-03-01

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

  6. Prominent features in isotopic, chemical and dust stratigraphies from GV7, a drilling site in East Antarctica

    NASA Astrophysics Data System (ADS)

    Caiazzo, Laura

    2016-04-01

    In the framework of the new project "The IPICS 2k Array: a network of ice core climate and climate forcing records for the last two millennia", which represents a thematic research line of International Partnerships in Ice Core Sciences (IPICS), a 250 m deep ice core was retrieved (spanning roughly the last millennium) at GV7 site, together with several shallow firn cores and snow pits. The PNRA (Programma Nazionale di Ricerche in Antartide) project "IPICS-2kyr-It" represents the Italian contribution to IPICS "The 2k Array" and it is being accomplished in collaboration with KOPRI (Korean Polar Reasearch Institute). The availability of various records from the same site all spanning a temporal period ranging from the last decades to the last centuries will allow achieving a stacked record of chemical and isotopic markers and accumulation rate that is basic for a reliable climatic reconstruction. Previous surveys in the area of GV7 (70°41' S - 158°51' E, 1950 m a.s.l., East Antarctica) showed that this site is characterized by a relatively high snow accumulation (about 240 mm water eq./year), allowing a high resolution study of the climatic variability in the last millennium. Here we present the isotopic, chemical and dust stratigraphies of the snow pits sampled at GV7 during the 2013/14 field season and analysed in Italy and in Korea. Reversibly deposited components such as nitrate and methansulphonic acid (MSA) appear to be well preserved and show a clear seasonal profiles, as one can observe from the records achieved both by Italian and Korean labs. Such a feature, together with the high accumulation rate, allowed obtaining an accurate dating of the snow pits, based on the counting of annual layers. At this purpose, a multi-parametric approach was chosen by using MSA, non-sea-salt sulphate, and d18O as seasonal markers. The dating confirmed the value of the accumulation rate found during previous samplings.

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

    SciTech Connect

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

    1992-02-01

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

  8. VARIABLE ACTIVE SITE LOOP CONFORMATIONS ACCOMMODATE THE BINDING OF MACROCYCLIC LARGAZOLE ANALOGUES TO HDAC8

    PubMed Central

    Decroos, Christophe; Clausen, Dane J.; Haines, Brandon E.; Wiest, Olaf; Williams, Robert M.; Christianson, David W.

    2015-01-01

    The macrocyclic depsipeptide Largazole is a potent inhibitor of metal-dependent histone deacetylases (HDACs), some of which are drug targets for cancer chemotherapy. Indeed, Largazole partially resembles Romidepsin (FK228), a macrocyclic depsipeptide already approved for clinical use. Each inhibitor contains a pendant side chain thiol that coordinates to the active site Zn2+ ion, as observed in the X-ray crystal structure of the HDAC8–Largazole complex [Cole, K. E.; Dowling, D. P.; Boone, M. A.; Phillips, A. J.; Christianson, D. W. J. Am. Chem. Soc. 2011, 133, 12474]. Here, we report the X-ray crystal structures of HDAC8 complexed with three synthetic analogues of Largazole in which the depsipeptide ester is replaced with a rigid amide linkage. In two of these analogues, a 6-membered pyridine ring is also substituted (with two different orientations) for the 5-membered thiazole ring in the macrocycle skeleton. The side chain thiol group of each analogue coordinates to the active site Zn2+ ion with nearly ideal geometry, thereby preserving the hallmark structural feature of inhibition by Largazole. Surprisingly, in comparison with the binding of Largazole, these analogues trigger alternative conformational changes in the L1 and L2 loops flanking the active site. However, despite these structural differences, inhibitory potency is generally comparable to, or just moderately less than, the inhibitory potency of Largazole. Thus, this study reveals important new structure-affinity relationships for the binding of macrocyclic inhibitors to HDAC8. PMID:25793284

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    2015-01-01

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

  12. Research on identification of active volcano features based on Landsat TM/ETM+ imagery

    NASA Astrophysics Data System (ADS)

    Kong, Xiangsheng; Qian, Yonggang

    2009-10-01

    Volcanic activity can present unpredictable disasters to city populations living within regions and for people traveling in plane that intersect with ash-laden eruption clouds. Methods of monitoring volcanic activity include searching for variations in the thermal anomaly, clouds resource and subsidence deformation from active volcano. Over any active volcanoes, low spatial resolution satellite image are used to identify changes in eruptive activity, but are of insufficient spatial resolution to map active volcanic features. The Landsat data can be used to identify the thermal characteristics of a series of lava flows at Fuego volcano and Pacaya volcano, Guatemala. We use Landsat TM/ETM+ 7, 5, 4 (displayed in red, green, and blue, respectively) false-color composite of the research region, acquired on 18 December 1989 and 23 January 2000 to indicate the volcano image features which appear halo structure with blue red and yellow. The interpretation flag is obvious which indicate the difference temperature of volcano crater. Spatially varying haze emitted by volcano activity is identified and removed based on Improved Haze Optimized Transform (HOT) which is a robust haze assessing method. With improved spatial resolution in the thermal IR, we are able to map the bifurcation and braiding of underground lava tubes. With higher spatial resolution panchromatic data, we are able to map lava flow fields, trace very high temperature lava channels, and identify an accurate feature associated with a collapsed crater floor. At both Fuego and Pacaya, we are able to use the thermal data to estimate temperature. We can monitor the dynamic change of the two volcanoes using two difference date Landsat data.

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

    PubMed

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

    2016-07-01

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

  14. Feature integration in visual working memory: parietal gamma activity is related to cognitive coordination.

    PubMed

    Morgan, Helen M; Muthukumaraswamy, Suresh D; Hibbs, Carina S; Shapiro, Kimron L; Bracewell, R Martyn; Singh, Krish D; Linden, David E J

    2011-12-01

    The mechanism by which distinct subprocesses in the brain are coordinated is a central conundrum of systems neuroscience. The parietal lobe is thought to play a key role in visual feature integration, and oscillatory activity in the gamma frequency range has been associated with perception of coherent objects and other tasks requiring neural coordination. Here, we examined the neural correlates of integrating mental representations in working memory and hypothesized that parietal gamma activity would be related to the success of cognitive coordination. Working memory is a classic example of a cognitive operation that requires the coordinated processing of different types of information and the contribution of multiple cognitive domains. Using magnetoencephalography (MEG), we report parietal activity in the high gamma (80-100 Hz) range during manipulation of visual and spatial information (colors and angles) in working memory. This parietal gamma activity was significantly higher during manipulation of visual-spatial conjunctions compared with single features. Furthermore, gamma activity correlated with successful performance during the conjunction task but not during the component tasks. Cortical gamma activity in parietal cortex may therefore play a role in cognitive coordination. PMID:21940605

  15. Perception of race-related features modulates neural activity associated with action observation and imitation.

    PubMed

    Earls, Holly A; Englander, Zoë A; Morris, James P

    2013-05-29

    The present study examines whether race-specific features affect biological motion perception. Activation of the neural action observation and imitation network was measured using functional MRI. During scanning, individuals were asked to imitate and observe basic hand movements of own-race and other-race actors. Results indicate that three key areas often associated with action observation and imitation, the inferior parietal lobule, superior parietal lobule, and superior temporal sulcus, were more active when participants imitated and observed hand movements of own-race relative to other-race actors. These findings indicate that several regions associated with the neural imitation/observation network are sensitive to race-related features. PMID:23571693

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

    PubMed

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

    2015-01-01

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

  17. In-silico analysis of binding site features and substrate selectivity in plant flavonoid-3-O glycosyltransferases (F3GT) through molecular modeling, docking and dynamics simulation studies.

    PubMed

    Sharma, Ranu; Panigrahi, Priyabrata; Suresh, C G

    2014-01-01

    Flavonoids are a class of plant secondary metabolites that act as storage molecules, chemical messengers, as well as participate in homeostasis and defense processes. They possess pharmaceutical properties important for cancer treatment such as antioxidant and anti-tumor activities. The drug-related properties of flavonoids can be improved by glycosylation. The enzymes glycosyltransferases (GTs) glycosylate acceptor molecules in a regiospecific manner with the help of nucleotide sugar donor molecules. Several plant GTs have been characterized and their amino acid sequences determined. However, three-dimensional structures of only a few are reported. Here, phylogenetic analysis using amino acid sequences have identified a group of GTs with the same regiospecific activity. The structures of these closely related GTs were modeled using homologous GT structures. Their substrate binding sites were elaborated by docking flavonoid acceptor and UDP-sugar donor molecules in the modeled structures. Eight regions near the acceptor binding site in the N- and C- terminal domain of GTs have been identified that bind and specifically glycosylate the 3-OH group of acceptor flavonoids. Similarly, a conserved motif in the C-terminal domain is known to bind a sugar donor substrate. In certain GTs, the substitution of a specific glutamine by histidine in this domain changes the preference of sugar from glucose to galactose as a result of changed pattern of interactions. The molecular modeling, docking, and molecular dynamics simulation studies have revealed the chemical and topological features of the binding site and thus provided insights into the basis of acceptor and donor recognition by GTs. PMID:24667893

  18. In-Silico Analysis of Binding Site Features and Substrate Selectivity in Plant Flavonoid-3-O Glycosyltransferases (F3GT) through Molecular Modeling, Docking and Dynamics Simulation Studies

    PubMed Central

    Sharma, Ranu; Panigrahi, Priyabrata; Suresh, C.G.

    2014-01-01

    Flavonoids are a class of plant secondary metabolites that act as storage molecules, chemical messengers, as well as participate in homeostasis and defense processes. They possess pharmaceutical properties important for cancer treatment such as antioxidant and anti-tumor activities. The drug-related properties of flavonoids can be improved by glycosylation. The enzymes glycosyltransferases (GTs) glycosylate acceptor molecules in a regiospecific manner with the help of nucleotide sugar donor molecules. Several plant GTs have been characterized and their amino acid sequences determined. However, three-dimensional structures of only a few are reported. Here, phylogenetic analysis using amino acid sequences have identified a group of GTs with the same regiospecific activity. The structures of these closely related GTs were modeled using homologous GT structures. Their substrate binding sites were elaborated by docking flavonoid acceptor and UDP-sugar donor molecules in the modeled structures. Eight regions near the acceptor binding site in the N- and C- terminal domain of GTs have been identified that bind and specifically glycosylate the 3-OH group of acceptor flavonoids. Similarly, a conserved motif in the C-terminal domain is known to bind a sugar donor substrate. In certain GTs, the substitution of a specific glutamine by histidine in this domain changes the preference of sugar from glucose to galactose as a result of changed pattern of interactions. The molecular modeling, docking, and molecular dynamics simulation studies have revealed the chemical and topological features of the binding site and thus provided insights into the basis of acceptor and donor recognition by GTs. PMID:24667893

  19. Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites

    PubMed Central

    Savic, Daniel; Roberts, Brian S.; Carleton, Julia B.; Partridge, E. Christopher; White, Michael A.; Cohen, Barak A.; Cooper, Gregory M.; Gertz, Jason; Myers, Richard M.

    2015-01-01

    Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type–specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type–specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites. PMID:26486725

  20. Lessons Learned from WIPP Site Characteriztion, Performance Assessment, and Regulatory Review Related to Radionuclide Migration through Water-Conducting Features

    SciTech Connect

    Beauheim, R.L.: Larson. K.W.

    1998-11-11

    Many lessons have been learned over the past 24 years as the Waste Isolation Pilot Plant (WIPP) project has progressed from initial site characterization to final licensing that may be of relevance to other nuclear-waste-disposal projects. These lessons pertain to the manner in which field and laboratory investigations are planned, how experiments are interpreted, how conceptual and numerical models are developed and simplified~ and how defensibility and credibility are achieved and maintained. These lessons include 1) Site characterization and performance assessment (PA) should evolve together through an iterative process, with neither activity completely dominating the other. 2) Defensibility and credibility require a much greater depth of understanding than can be represented in PA models. 3) Experimentalists should be directly involved in model and parameter abstraction and simplification for PA. 4) External expert review should be incorporated at all stages of a project~ not just after an experiment or modeling activity is completed. 5) Key individuals should be retained for the life of a project or a process must be established to transfer their working knowledge to new individuals. 6) An effective QA program needs to be stable and consistent for the duration of a project and rests on best scientific practices. All of these lessons relate to the key point that consideration must be given from the earliest planning stages to maximizing the defensibility and credibility of all work.

  1. Clinical features of active tuberculosis that developed during anti-tumor necrosis factor therapy in patients with inflammatory bowel disease

    PubMed Central

    Lee, Jang Wook; Park, Ji Hoon; Kim, Jeong Wook; Kang, Sang Bum; Koo, Ja Seol; Kim, Young-Ho; Kim, You Sun; Joo, Young Eun; Chang, Sae Kyung

    2016-01-01

    Background/Aims Anti-tumor necrosis factor (TNF) therapy for active ulcerative colitis (UC) and Crohn's disease (CD) is associated with increased risks of tuberculosis (TB) infection. We analyzed the incidence and clinical features of Korean patients with inflammatory bowel disease (IBD) who developed active TB during anti-TNF therapy. Methods Ten cases of active TB developed in patients treated with infliximab (n=592) or adalimumab (n=229) for UC (n=160) or CD (n=661) were reviewed. We analyzed demographics, interval between start of anti-TNF therapy and active TB development, tests for latent TB infection (LTBI), concomitant medications, and the details of diagnosis and treatments for TB. Results The incidence of active TB was 1.2% (10/821): 1.5% (9/592) and 0.4% (1/229) in patients receiving infliximab and adalimumab, respectively. The median time to the development of active TB after initiation of anti-TNF therapy was three months (range: 2–36). Three patients had past histories of treatment for TB. Positive findings in a TB skin test (TST) and/or interferon gamma releasing assay (IGRA) were observed in three patients, and two of them received anti-TB prophylaxis. Two patients were negative by both TST and IGRA. The most common site of active TB was the lungs, and the active TB was cured in all patients. Conclusions Active TB can develop during anti-TNF therapy in IBD patients without LTBI, and even in those with histories of TB treatment or LTBI prophylaxis. Physicians should be aware of the potential for TB development during anti-TNF therapy, especially in countries with a high prevalence of TB. PMID:27175115

  2. Analysis of Hydrogen Tunneling in an Enzyme Active Site using von Neumann Measurements

    PubMed Central

    Sumner, Isaiah; Iyengar, Srinivasan S.

    2010-01-01

    We build on our earlier quantum wavepacket study of hydrogen transfer in the biological enzyme, soybean lipoxygenase-1, by using von Neumann quantum measurement theory to gain qualitative insights into the transfer event. We treat the enzyme active site as a measurement device which acts on the tunneling hydrogen nucleus via the potential it exerts at each configuration. A series of changing active site geometries during the tunneling process effects a sequential projection of the initial, reactant state onto the final, product state. We study this process using several different kinds of von Neumann measurements and show how a discrete sequence of such measurements not only progressively increases the projection of the hydrogen nuclear wavepacket onto the product side but also favors proton over deuteron transfer. Several qualitative features of the hydrogen tunneling problem found in wavepacket dynamics studies are also recovered here. These include the shift in the “transition state” towards the reactant as a result of nuclear quantization, greater participation of excited states in the case of deuterium, and presence of critical points along the reaction coordinate that facilitate hydrogen and deuterium transfer and coincide with surface crossings. To further “tailor” the dynamics, we construct a perturbation to the sequence of measurements, that is a perturbation to the dynamical sequence of active site geometry evolution, which leads us to insight on the existence of sensitive regions of the reaction profile where subtle changes to the dynamics of the active site can have an effect on the hydrogen and deuterium transfer process. PMID:22933858

  3. Optimization studies on the features of an activated charcoal-supported urease system.

    PubMed

    Kibarer, G D; Akovali, G

    1996-08-01

    The adsorption of urease onto a well-defined solid support, petroleum-based activated charcoal, has been achieved to provide the enzymatic hydrolysis of urea. In order to produce a biocompatible surface, the enzyme support system has been coated with hexamethyldisiloxane through plasma polymerization. The quality of the resulting coat was tested by electronic spectroscopy for chemical analysis and scanning electron microscopy techniques. Studies on the adsorption of urease, and activity and stability of the enzyme on the support have been in the direction to optimize the features of the charcoal-supported urease and improve its availability for further use in clinical applications. PMID:8853117

  4. Characteristic Tandem Mass Spectral Features Under Various Collision Chemistries for Site-Specific Identification of Protein S-Glutathionylation

    NASA Astrophysics Data System (ADS)

    Chou, Chi-Chi; Chiang, Bing-Yu; Lin, Jason Ching-Yao; Pan, Kuan-Ting; Lin, Chun-Hung; Khoo, Kay-Hooi

    2015-01-01

    Protein S-glutathionylation is a reversible post-translational modification widely implicated in redox regulated biological functions. Conventional biochemical methods, however, often do not allow such a mixed disulfide modification to be reliably identified on specific cysteine residues or be distinguished from other related oxidized forms. To develop more efficient mass spectrometry (MS)-based analytical strategies for this purpose, we first investigated the MS/MS fragmentation pattern of S-glutathionylated peptides under various dissociation modes, including collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), and electron transfer dissociation (ETD), using synthetic peptides derived from protein tyrosine phosphatase as models. Our results indicate that a MALDI-based high energy CID MS/MS on a TOF/TOF affords the most distinctive spectral features that would facilitate rapid and unambiguous identification of site-specific S-glutathionylation. For more complex proteomic samples best tackled by LC-MS/MS approach, we demonstrate that HCD performed on an LTQ-Orbitrap hybrid instrument fairs better than trap-based CID and ETD in allowing more protein site-specific S-glutathionylation to be confidently identified by direct database searching of the generated MS/MS dataset using Mascot. Overall, HCD afforded more peptide sequence-informative fragment ions retaining the glutathionyl modification with less neutral losses of side chains to compromise scoring. In conjunction with our recently developed chemo-enzymatic tagging strategy, our nanoLC-HCD-MS/MS approach is sufficiently sensitive to identify endogenous S-glutathionylated peptides prepared from non-stressed cells. It is anticipated that future applications to global scale analysis of protein S-glutathionylation will benefit further from current advances in both speed and mass accuracy afforded by HCD MS/MS mode on the Orbitrap series.

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

    PubMed

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

    2012-09-01

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

  6. Structural and conformational features relevant to the anti-tumor activity of calicheamicin γ 1I.

    PubMed

    Ellestad, George A

    2011-09-01

    The structural and conformational features of the potent 10-membered enediyne-containing calicheamicin γ 1I that account for its remarkable DNA site-specific binding and cleavage are reviewed. A variety of spectroscopic and biophysical techniques were used to gain insight into the binding and stereospecific DNA cleavage of this potent antitumor agent. These include gel-shift cleavage assays, atom transfer NMR experiments, drug-DNA conformational studies, circular dichroism, and capillary electrophoresis. Computational descriptions are described for the DNA binding and cleavage of calicheamicin and its activated transient intermediates based on density functional and molecular mechanics calculations. In addition, the structure and clinical utility of calicheamicin immunoconjugates for antibody-targeted chemotherapy is presented. PMID:21800378

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

    PubMed

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

    2015-07-21

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

  8. RNA-Seq of Bacillus licheniformis: active regulatory RNA features expressed within a productive fermentation

    PubMed Central

    2013-01-01

    Background The production of enzymes by an industrial strain requires a complex adaption of the bacterial metabolism to the conditions within the fermenter. Regulatory events within the process result in a dynamic change of the transcriptional activity of the genome. This complex network of genes is orchestrated by proteins as well as regulatory RNA elements. Here we present an RNA-Seq based study considering selected phases of an industry-oriented fermentation of Bacillus licheniformis. Results A detailed analysis of 20 strand-specific RNA-Seq datasets revealed a multitude of transcriptionally active genomic regions. 3314 RNA features encoded by such active loci have been identified and sorted into ten functional classes. The identified sequences include the expected RNA features like housekeeping sRNAs, metabolic riboswitches and RNA switches well known from studies on Bacillus subtilis as well as a multitude of completely new candidates for regulatory RNAs. An unexpectedly high number of 855 RNA features are encoded antisense to annotated protein and RNA genes, in addition to 461 independently transcribed small RNAs. These antisense transcripts contain molecules with a remarkable size range variation from 38 to 6348 base pairs in length. The genome of the type strain B. licheniformis DSM13 was completely reannotated using data obtained from RNA-Seq analyses and from public databases. Conclusion The hereby generated data-sets represent a solid amount of knowledge on the dynamic transcriptional activities during the investigated fermentation stages. The identified regulatory elements enable research on the understanding and the optimization of crucial metabolic activities during a productive fermentation of Bacillus licheniformis strains. PMID:24079885

  9. Relocating the active-site lysine in rhodopsin and implications for evolution of retinylidene proteins

    PubMed Central

    Devine, Erin L.; Oprian, Daniel D.; Theobald, Douglas L.

    2013-01-01

    Type I and type II rhodopsins share several structural features including a G protein-coupled receptor fold and a highly conserved active-site Lys residue in the seventh transmembrane segment of the protein. However, the two families lack significant sequence similarity that would indicate common ancestry. Consequently, the rhodopsin fold and conserved Lys are widely thought to have arisen from functional constraints during convergent evolution. To test for the existence of such a constraint, we asked whether it were possible to relocate the highly conserved Lys296 in the visual pigment bovine rhodopsin. We show here that the Lys can be moved to three other locations in the protein while maintaining the ability to form a pigment with 11-cis-retinal and activate the G protein transducin in a light-dependent manner. These results contradict the convergent hypothesis and support the homology of type I and type II rhodopsins by divergent evolution from a common ancestral protein. PMID:23904486

  10. Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

    PubMed Central

    Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

    2008-01-01

    The absorption spectra of N-acetyl-L-tryptophanamide in various solvents were resolved into the sums of the 1La and 1Lb components. The relative intensities of the 0-0 transitions of the 1Lb bands correlate linearly with the solvent polarity values (ETN). A novel strategy, which utilizes a set of the experimental 1Lb bands, was employed to resolve the near-UV CD spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence as well as structural data of TL. Analysis of the 1Lb bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The “DMSO-like” 1Lb band of Trp CD spectra may be used as a “fingerprint” to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side-chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method we call site-directed circular dichroism is broadly applicable to various proteins to obtain the position-specific data. PMID:18047823

  11. Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2008-03-15

    The absorption spectra of N-acetyl-L-tryptophanamide in various solvents were resolved into the sums of the (1)L(a) and (1)L(b) components. The relative intensities of the 0-0 transitions of the (1)L(b) bands correlate linearly with the solvent polarity values (E(T)(N)). A novel strategy that uses a set of the experimental (1)L(b) bands was employed to resolve the near-UV circular dichroism (CD) spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence and structural data of TL. Analysis of the (1)L(b) bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The dimethyl sulfoxide (DMSO)-like (1)L(b) band of Trp CD spectra may be used as a "fingerprint" to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method, which we call site-directed circular dichroism, is broadly applicable to various proteins to obtain the position-specific data. PMID:18047823

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

    PubMed

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

    2015-02-24

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

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

    PubMed

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

    2015-05-01

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

  14. Feature extraction and recognition of epileptiform activity in EEG by combining PCA with ApEn.

    PubMed

    Wang, Chunmei; Zou, Junzhong; Zhang, Jian; Wang, Min; Wang, Rubin

    2010-09-01

    This paper proposes a new method for feature extraction and recognition of epileptiform activity in EEG signals. The method improves feature extraction speed of epileptiform activity without reducing recognition rate. Firstly, Principal component analysis (PCA) is applied to the original EEG for dimension reduction and to the decorrelation of epileptic EEG and normal EEG. Then discrete wavelet transform (DWT) combined with approximate entropy (ApEn) is performed on epileptic EEG and normal EEG, respectively. At last, Neyman-Pearson criteria are applied to classify epileptic EEG and normal ones. The main procedure is that the principle component of EEG after PCA is decomposed into several sub-band signals using DWT, and ApEn algorithm is applied to the sub-band signals at different wavelet scales. Distinct difference is found between the ApEn values of epileptic and normal EEG. The method allows recognition of epileptiform activities and discriminates them from the normal EEG. The algorithm performs well at epileptiform activity recognition in the clinic EEG data and offers a flexible tool that is intended to be generalized to the simultaneous recognition of many waveforms in EEG. PMID:21886676

  15. Feature extraction and recognition of epileptiform activity in EEG by combining PCA with ApEn

    PubMed Central

    Zou, Junzhong; Zhang, Jian; Wang, Min; Wang, Rubin

    2010-01-01

    This paper proposes a new method for feature extraction and recognition of epileptiform activity in EEG signals. The method improves feature extraction speed of epileptiform activity without reducing recognition rate. Firstly, Principal component analysis (PCA) is applied to the original EEG for dimension reduction and to the decorrelation of epileptic EEG and normal EEG. Then discrete wavelet transform (DWT) combined with approximate entropy (ApEn) is performed on epileptic EEG and normal EEG, respectively. At last, Neyman–Pearson criteria are applied to classify epileptic EEG and normal ones. The main procedure is that the principle component of EEG after PCA is decomposed into several sub-band signals using DWT, and ApEn algorithm is applied to the sub-band signals at different wavelet scales. Distinct difference is found between the ApEn values of epileptic and normal EEG. The method allows recognition of epileptiform activities and discriminates them from the normal EEG. The algorithm performs well at epileptiform activity recognition in the clinic EEG data and offers a flexible tool that is intended to be generalized to the simultaneous recognition of many waveforms in EEG. PMID:21886676

  16. Geomorphological features of active tectonics and ongoing seismicity of northeastern Kumaun Himalaya, Uttarakhand, India

    NASA Astrophysics Data System (ADS)

    Pathak, Vivekanand; Pant, Charu C.; Darmwal, Gopal Singh

    2015-08-01

    The northeastern part of Kumaun Lesser Himalaya, Uttarakhand, India, lying between the rupture zones of 1905, Kangra and 1934, Bihar-Nepal earthquakes and known as `central seismic gap' is a segment of an active fault known to produce significant earthquakes and has not slipped in an unusually long time when compared to other segments. The studied section forms a part of this seismic gap and is seismically an active segment of the Himalayan arc, as compared to the remaining part of the Kumaun Lesser Himalaya and it is evident by active geomorphological features and seismicity data. The geomorphological features of various river valley transects suggest that the region had a history of tectonic rejuvenation which is testified by the deposition of various levels of terraces and their relative uplift, shifting and ponding of river channels, uplifted potholes, triangular facets on fault planes, fault scarps, etc. Further, the seismic data of five-station digital telemetered seismic network along with two stand alone systems show the distribution of earthquakes in or along the analyzed fault transects. It is observed that the microseismic earthquakes (magnitude 1.0-3.0) frequently occur in the region and hypocenters of these earthquakes are confined to shallow depths (10-20 km), with low stress drop values (1.0-10 bar) and higher peak ground velocity (PGV). The cluster of events is observed in the region, sandwiched between the Berinag Thrust (BT) in south and Main Central Thrust (MCT) in north. The occurrences of shallow focus earthquakes and the surface deformational features in the different river valley transect indicates that the region is undergoing neotectonic rejuvenation. In absence of chronology of the deposits it is difficult to relate it with extant seismicity, but from the geomorphic and seismic observations it may be concluded that the region is still tectonically active. The information would be very important in identifying the areas of hazard prone and

  17. Identifying Key Features of Effective Active Learning: The Effects of Writing and Peer Discussion

    PubMed Central

    Pangle, Wiline M.; Wyatt, Kevin H.; Powell, Karli N.; Sherwood, Rachel E.

    2014-01-01

    We investigated some of the key features of effective active learning by comparing the outcomes of three different methods of implementing active-learning exercises in a majors introductory biology course. Students completed activities in one of three treatments: discussion, writing, and discussion + writing. Treatments were rotated weekly between three sections taught by three different instructors in a full factorial design. The data set was analyzed by generalized linear mixed-effect models with three independent variables: student aptitude, treatment, and instructor, and three dependent (assessment) variables: change in score on pre- and postactivity clicker questions, and coding scores on in-class writing and exam essays. All independent variables had significant effects on student performance for at least one of the dependent variables. Students with higher aptitude scored higher on all assessments. Student scores were higher on exam essay questions when the activity was implemented with a writing component compared with peer discussion only. There was a significant effect of instructor, with instructors showing different degrees of effectiveness with active-learning techniques. We suggest that individual writing should be implemented as part of active learning whenever possible and that instructors may need training and practice to become effective with active learning. PMID:25185230

  18. Identifying key features of effective active learning: the effects of writing and peer discussion.

    PubMed

    Linton, Debra L; Pangle, Wiline M; Wyatt, Kevin H; Powell, Karli N; Sherwood, Rachel E

    2014-01-01

    We investigated some of the key features of effective active learning by comparing the outcomes of three different methods of implementing active-learning exercises in a majors introductory biology course. Students completed activities in one of three treatments: discussion, writing, and discussion + writing. Treatments were rotated weekly between three sections taught by three different instructors in a full factorial design. The data set was analyzed by generalized linear mixed-effect models with three independent variables: student aptitude, treatment, and instructor, and three dependent (assessment) variables: change in score on pre- and postactivity clicker questions, and coding scores on in-class writing and exam essays. All independent variables had significant effects on student performance for at least one of the dependent variables. Students with higher aptitude scored higher on all assessments. Student scores were higher on exam essay questions when the activity was implemented with a writing component compared with peer discussion only. There was a significant effect of instructor, with instructors showing different degrees of effectiveness with active-learning techniques. We suggest that individual writing should be implemented as part of active learning whenever possible and that instructors may need training and practice to become effective with active learning. PMID:25185230

  19. Comparison of the surface features of two wood-based activated carbons

    SciTech Connect

    Salame, I.I.; Bandosz, T.J.

    2000-02-01

    The surface features of two carbons of wood origin were compared. One sample was manufactured using phosphoric acid activation and the other using potassium hydroxide activation. To check the susceptibility to oxidation and the stability of the porous structure, the samples were oxidized with ammonium persulfate. Structural properties of carbons and their oxidized counterparts were determined using sorption of nitrogen. Surface acidity was evaluated using Boehm titration, potentiometric titration, inverse gas chromatography, and diffuse reflectance FTIR. It was demonstrated that, despite the same wood origin, the carbons significantly differ in their pore structure and surface chemistry. The carbon obtained using KOH activation is homogeneously microporous with high surface area around 2,300 m{sup 2}/g (BET). On the other hand, the carbon manufactured using phosphoric acid contains a high volume of mesopores and its surface area is significantly lower. The carbons also differ in their surface chemistry and susceptibility to oxidation.

  20. Distinctive microstructural features of aged sodium silicate-activated slag concretes

    SciTech Connect

    San Nicolas, Rackel; Bernal, Susan A.; Mejía de Gutiérrez, Ruby; Deventer, Jannie S.J. van; Provis, John L.

    2014-11-15

    Electron microscopic characterisation of 7-year old alkali-activated blast-furnace slag concretes enabled the identification of distinct microstructural features, providing insight into the mechanisms by which these materials evolve over time. Backscattered electron images show the formation of Liesegang-type ring formations, suggesting that the reaction at advanced age is likely to follow an Oswald supersaturation–nucleation–depletion cycle. Segregation of Ca-rich veins, related to the formation of Ca(OH){sub 2}, is observed in microcracked regions due to the ongoing reaction between the pore solution and available calcium from remnant slag grains. A highly dense and uniform interfacial transition zone is identified between siliceous aggregate particles and the alkali activated slag binders, across the concretes assessed. Alkali-activated slag concretes retain a highly dense and stable microstructure at advanced ages, where any microcracks induced at early ages seem to be partially closing, and the remnant slag grains continue reacting.

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

    PubMed

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

    2016-08-01

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

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

    PubMed

    Li, Chi-Hui; Tu, Shiao-Chun

    2005-10-01

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

  3. Music-induced emotions can be predicted from a combination of brain activity and acoustic features.

    PubMed

    Daly, Ian; Williams, Duncan; Hallowell, James; Hwang, Faustina; Kirke, Alexis; Malik, Asad; Weaver, James; Miranda, Eduardo; Nasuto, Slawomir J

    2015-12-01

    It is widely acknowledged that music can communicate and induce a wide range of emotions in the listener. However, music is a highly-complex audio signal composed of a wide range of complex time- and frequency-varying components. Additionally, music-induced emotions are known to differ greatly between listeners. Therefore, it is not immediately clear what emotions will be induced in a given individual by a piece of music. We attempt to predict the music-induced emotional response in a listener by measuring the activity in the listeners electroencephalogram (EEG). We combine these measures with acoustic descriptors of the music, an approach that allows us to consider music as a complex set of time-varying acoustic features, independently of any specific music theory. Regression models are found which allow us to predict the music-induced emotions of our participants with a correlation between the actual and predicted responses of up to r=0.234,p<0.001. This regression fit suggests that over 20% of the variance of the participant's music induced emotions can be predicted by their neural activity and the properties of the music. Given the large amount of noise, non-stationarity, and non-linearity in both EEG and music, this is an encouraging result. Additionally, the combination of measures of brain activity and acoustic features describing the music played to our participants allows us to predict music-induced emotions with significantly higher accuracies than either feature type alone (p<0.01). PMID:26544602

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

    PubMed

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

    2014-06-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    1982-01-01

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

  7. Characterization of active site residues of nitroalkane oxidase.

    PubMed

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

    2010-06-01

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

  8. Detection limit for activation measurements in ultralow background sites

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. A comparative structure-function analysis of active-site inhibitors of Vibrio cholerae cholix toxin.

    PubMed

    Lugo, Miguel R; Merrill, A Rod

    2015-09-01

    Cholix toxin from Vibrio cholerae is a novel mono-ADP-ribosyltransferase (mART) toxin that shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin. Herein, we have used the high-resolution X-ray structure of full-length cholix toxin in the apo form, NAD(+) bound, and 10 structures of the cholix catalytic domain (C-domain) complexed with several strong inhibitors of toxin enzyme activity (NAP, PJ34, and the P-series) to study the binding mode of the ligands. A pharmacophore model based on the active pose of NAD(+) was compared with the active conformation of the inhibitors, which revealed a cationic feature in the side chain of the inhibitors that may determine the active pose. Moreover, a conformational search was conducted for the missing coordinates of one of the main active-site loops (R-loop). The resulting structural models were used to evaluate the interaction energies and for 3D-QSAR modeling. Implications for a rational drug design approach for mART toxins were derived. PMID:25756608

  11. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

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

    Code of Federal Regulations, 2014 CFR

    2014-01-01

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

  13. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2012 CFR

    2012-01-01

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

  14. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2013 CFR

    2013-01-01

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

  15. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2011 CFR

    2011-01-01

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

  16. Monocyte activation is a feature of common variable immunodeficiency irrespective of plasma lipopolysaccharide levels

    PubMed Central

    Barbosa, R R; Silva, S P; Silva, S L; Tendeiro, R; Melo, A C; Pedro, E; Barbosa, M P; Santos, M C P; Victorino, R M M; Sousa, A E

    2012-01-01

    Common variable immunodeficiency disorders (CVID), the most frequent cause of symptomatic primary immunodeficiency, are defined by impaired antibody production. Notwithstanding, T cell activation and granulomatous manifestations represent the main causes of CVID morbidity even in patients receiving immunoglobulin (Ig) G replacement therapy. Additionally, gut pathology is a frequent feature of CVID. In this study, we investigated monocyte imbalances and their possible relationship with increased microbial translocation in CVID patients. Monocyte subsets were defined according to CD14 and CD16 expression levels and evaluated in terms of human leucocyte antigen D-related (HLA-DR), CD86 and programmed death-1 molecule ligand 1 (PD-L1) expression by flow cytometry, in parallel with the quantification of plasma lipopolysaccharide (LPS) and serum levels of soluble CD14 (sCD14), LPS-binding protein (LBP) and anti-LPS antibodies. CVID patients (n = 31) featured significantly increased levels of serum sCD14 and an expansion of CD14brightCD16+ monocytes in direct correlation with T cell and B cell activation, the latter illustrated by the frequency of the CD21lowCD38low subset. Such alterations were not observed in patients lacking B cells due to congenital agammaglobulinaemia (n = 4). Moreover, we found no significant increase in circulating LPS or LBP levels in CVID patients, together with a relative preservation of serum anti-LPS antibodies, in agreement with their presence in commercial IgG preparations. In conclusion, CVID was associated with monocyte imbalances that correlated directly with T cell activation markers and with B cell imbalances, without an association with plasma LPS levels. The heightened monocyte activated state observed in CVID may represent an important target for complementary therapeutic strategies. PMID:22861366

  17. The energy balance and pressure in the solar transition zone for network and active region features

    NASA Technical Reports Server (NTRS)

    Nicolas, K. R.; Bartoe, J.-D. F.; Brueckner, G. E.; Vanhoosier, M. E.

    1979-01-01

    The electron pressure and energy balance in the solar transition zone are determined for about 125 network and active region features on the basis of high spectral and spatial resolution extreme ultraviolet spectra. Si III line intensity ratios obtained from the Naval Research Laboratory high-resolution telescope and spectrograph during a rocket flight are used as diagnostics of electron density and pressure for solar features near 3.5 x 10 to the 4th K. Observed ratios are compared with the calculated dependence of the 1301 A/1312 A and 1301 A/1296 A line intensity ratios on electron density, temperature and pressure. Electron densities ranging from 2 x 10 to the 10th/cu cm to 10 to the 12th/cu cm and active region pressures from 3 x 10 to the 15th to 10 to the 16th/cu cm K are obtained. Energy balance calculations reveal the balance of the divergence of the conductive flux and turbulent energy dissipation by radiative energy losses in a plane-parallel homogeneous transition zone (fill factor of 1), and an energy source requirement for a cylindrical zone geometry (fill factor less than 0.04).

  18. THE INTRINSIC VALUE OF HFO FEATURES AS A BIOMARKER OF EPILEPTIC ACTIVITY

    PubMed Central

    Gliske, Stephen V.; Stacey, William C.; Moon, Kevin R.; Hero, Alfred O.

    2016-01-01

    High frequency oscillations (HFOs) are a promising biomarker of epileptic brain tissue and activity. HFOs additionally serve as a prototypical example of challenges in the analysis of discrete events in high-temporal resolution, intracranial EEG data. Two primary challenges are 1) dimensionality reduction, and 2) assessing feasibility of classification. Dimensionality reduction assumes that the data lie on a manifold with dimension less than that of the features space. However, previous HFO analysis have assumed a linear manifold, global across time, space (i.e. recording electrode/channel), and individual patients. Instead, we assess both a) whether linear methods are appropriate and b) the consistency of the manifold across time, space, and patients. We also estimate bounds on the Bayes classification error to quantify the distinction between two classes of HFOs (those occurring during seizures and those occurring due to other processes). This analysis provides the foundation for future clinical use of HFO features and guides the analysis for other discrete events, such as individual action potentials or multi-unit activity.

  19. Genomic features of uncultured methylotrophs in activated-sludge microbiomes grown under different enrichment procedures

    PubMed Central

    Fujinawa, Kazuki; Asai, Yusuke; Miyahara, Morio; Kouzuma, Atsushi; Abe, Takashi; Watanabe, Kazuya

    2016-01-01

    Methylotrophs are organisms that are able to grow on C1 compounds as carbon and energy sources. They play important roles in the global carbon cycle and contribute largely to industrial wastewater treatment. To identify and characterize methylotrophs that are involved in methanol degradation in wastewater-treatment plants, methanol-fed activated-sludge (MAS) microbiomes were subjected to phylogenetic and metagenomic analyses, and genomic features of dominant methylotrophs in MAS were compared with those preferentially grown in laboratory enrichment cultures (LECs). These analyses consistently indicate that Hyphomicrobium plays important roles in MAS, while Methylophilus occurred predominantly in LECs. Comparative analyses of bin genomes reconstructed for the Hyphomicrobium and Methylophilus methylotrophs suggest that they have different C1-assimilation pathways. In addition, function-module analyses suggest that their cell-surface structures are different. Comparison of the MAS bin genome with genomes of closely related Hyphomicrobium isolates suggests that genes unnecessary in MAS (for instance, genes for anaerobic respiration) have been lost from the genome of the dominant methylotroph. We suggest that genomic features and coded functions in the MAS bin genome provide us with insights into how this methylotroph adapts to activated-sludge ecosystems. PMID:27221669

  20. Genomic features of uncultured methylotrophs in activated-sludge microbiomes grown under different enrichment procedures.

    PubMed

    Fujinawa, Kazuki; Asai, Yusuke; Miyahara, Morio; Kouzuma, Atsushi; Abe, Takashi; Watanabe, Kazuya

    2016-01-01

    Methylotrophs are organisms that are able to grow on C1 compounds as carbon and energy sources. They play important roles in the global carbon cycle and contribute largely to industrial wastewater treatment. To identify and characterize methylotrophs that are involved in methanol degradation in wastewater-treatment plants, methanol-fed activated-sludge (MAS) microbiomes were subjected to phylogenetic and metagenomic analyses, and genomic features of dominant methylotrophs in MAS were compared with those preferentially grown in laboratory enrichment cultures (LECs). These analyses consistently indicate that Hyphomicrobium plays important roles in MAS, while Methylophilus occurred predominantly in LECs. Comparative analyses of bin genomes reconstructed for the Hyphomicrobium and Methylophilus methylotrophs suggest that they have different C1-assimilation pathways. In addition, function-module analyses suggest that their cell-surface structures are different. Comparison of the MAS bin genome with genomes of closely related Hyphomicrobium isolates suggests that genes unnecessary in MAS (for instance, genes for anaerobic respiration) have been lost from the genome of the dominant methylotroph. We suggest that genomic features and coded functions in the MAS bin genome provide us with insights into how this methylotroph adapts to activated-sludge ecosystems. PMID:27221669

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

    SciTech Connect

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

    2012-02-13

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

  2. Covalent Inhibition of Ubc13 Affects Ubiquitin Signaling and Reveals Active Site Elements Important for Targeting

    PubMed Central

    Hodge, Curtis D.; Edwards, Ross A.; Markin, Craig J.; McDonald, Darin; Pulvino, Mary; Huen, Michael S. Y.; Zhao, Jiyong; Spyracopoulos, Leo; Hendzel, Michael J.; Glover, J.N. Mark

    2015-01-01

    Ubc13 is an E2 ubiquitin conjugating enzyme that functions in nuclear DNA damage signaling and cytoplasmic NF-κB signaling. Here we present the structures of complexes of Ubc13 with two inhibitors, NSC697923 and BAY 11-7082, which inhibit DNA damage and NF-κB signaling in human cells. NSC697923 and BAY 11-7082 both inhibit Ubc13 by covalent adduct formation through a Michael addition at the Ubc13 active site cysteine. The resulting adducts of both compounds exploit a binding groove unique to Ubc13. We developed a Ubc13 mutant which resists NSC697923 inhibition and, using this mutant, we show that the inhibition of cellular DNA damage and NF-κB signaling by NSC697923 is largely due to specific Ubc13 inhibition. We propose that unique structural features near the Ubc13 active site could provide a basis for the rational development and design of specific Ubc13 inhibitors. PMID:25909880

  3. Covalent Inhibition of Ubc13 Affects Ubiquitin Signaling and Reveals Active Site Elements Important for Targeting.

    PubMed

    Hodge, Curtis D; Edwards, Ross A; Markin, Craig J; McDonald, Darin; Pulvino, Mary; Huen, Michael S Y; Zhao, Jiyong; Spyracopoulos, Leo; Hendzel, Michael J; Glover, J N Mark

    2015-07-17

    Ubc13 is an E2 ubiquitin conjugating enzyme that functions in nuclear DNA damage signaling and cytoplasmic NF-κB signaling. Here, we present the structures of complexes of Ubc13 with two inhibitors, NSC697923 and BAY 11-7082, which inhibit DNA damage and NF-κB signaling in human cells. NSC697923 and BAY 11-7082 both inhibit Ubc13 by covalent adduct formation through a Michael addition at the Ubc13 active site cysteine. The resulting adducts of both compounds exploit a binding groove unique to Ubc13. We developed a Ubc13 mutant which resists NSC697923 inhibition and, using this mutant, we show that the inhibition of cellular DNA damage and NF-κB signaling by NSC697923 is largely due to specific Ubc13 inhibition. We propose that unique structural features near the Ubc13 active site could provide a basis for the rational development and design of specific Ubc13 inhibitors. PMID:25909880

  4. QM/MM Analysis of Cellulase Active Sites and Actions of the Enzymes on Substrates

    SciTech Connect

    Saharay, Moumita; Guo, Hao-Bo; Smith, Jeremy C; Guo, Hong

    2010-01-01

    Biodegradation of cellulosic biomass requires the actions of three types of secreted enzymes; endoglucanase (EC 3.2.1.4), cellobiohydrolase or exoglucanase (EC 3.2.1.91), and -glucosidase (EC 4.2.1.21). These enzymes act synergistically to hydrolyse the -1,4 bonds of cellulose and converts it into simple sugar. Hydrolysis of the glycosidic bond can occur either by net retention or by inversion of anomeric configuration at the anomeric center. QM/MM simulations are useful tools to study the energetics of the reactions and analyze the active-site structures at different states of the catalysis, including the formation of unstable transition states. Here, a brief description of previous work on glycoside hydrolases is first given. The results of the QM/MM potential energy and free energy simulations corresponding to glycosylation and deglycosylation processes are then provided for two retaining endoglucanases, Cel12A and Cel5A. The active-site structural features are analyzed based on the QM/MM results. The role of different residues and hydrogen bonding interactions during the catalysis and the importance of the sugar ring distortion are discussed for these two enzymes.

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

    PubMed

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

    2001-08-01

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

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

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

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

  7. Dynamically Achieved Active Site Precision in Enzyme Catalysis

    PubMed Central

    2015-01-01

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

  8. Contribution of active-site glutamine to rate enhancement in ubiquitin carboxy terminal hydrolases

    PubMed Central

    Boudreaux, David; Chaney, Joseph; Maiti, Tushar K.; Das, Chittaranjan

    2012-01-01

    Ubiquitin carboxy terminal hydrolases (UCHs) are cysteine proteases featuring a classical cysteine-histidine-aspartate catalytic triad, also a highly conserved glutamine thought to be a part of the oxyanion hole. However, the contribution of this side chain to the catalysis by UCH enzymes is not known. Herein, we demonstrate that the glutamine side chain contributes to rate enhancement in UCHL1, UCHL3 and UCHL5. Mutation of the glutamine to alanine in these enzymes impairs the catalytic efficiency mainly due to a 16 to 30-fold reduction in kcat, which is consistent with a loss of approximately 2 kcal/mol in transition-state stabilization. However, the contribution to transition-state stabilization observed here is rather modest for the side chain’s role in oxyanion stabilization. Interestingly, we discovered that the carbonyl oxygen of this side chain is engaged in a C—H•••O hydrogen-bonding contact with the CεH group of the catalytic histidine. Upon further analysis, we found that this interaction is a common active-site structural feature in most cysteine proteases, including papain, belonging to families with the QCH(N/D) type of active-site configuration. It is possible that removal of the glutamine side chain might have abolished the C—H•••O interaction, which typically accounts for 2 kcal/mol of stabilization, leading to the effect on catalysis observed here. Additional studies performed on UCHL3 by mutating the glutamine to glutamate (strong C—H•••O acceptor but oxyanion destabilizer) and to lysine (strong oxyanion stabilizer but lacking C—H•••O hydrogen-bonding property) suggest that the C—H•••O hydrogen bond could contribute to catalysis. PMID:22284438

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

    SciTech Connect

    Teese, G.D.

    1995-09-28

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

  10. Frequencies, Laboratory Features, and Granulocyte Activation in Chinese Patients with CALR-Mutated Myeloproliferative Neoplasms

    PubMed Central

    Tian, Ruiyuan; Chang, Jianmei; Li, Jianlan; Tan, Yanhong; Xu, Zhifang; Ren, Fanggang; Zhao, Junxia; Pan, Jie; Zhang, Na; Wang, Xiaojuan; He, Jianxia; Yang, Wanfang; Wang, Hongwei

    2015-01-01

    Somatic mutations in the CALR gene have been recently identified as acquired alterations in myeloproliferative neoplasms (MPNs). In this study, we evaluated mutation frequencies, laboratory features, and granulocyte activation in Chinese patients with MPNs. A combination of qualitative allele-specific polymerase chain reaction and Sanger sequencing was used to detect three driver mutations (i.e., CALR, JAK2V617F, and MPL). CALR mutations were identified in 8.4% of cases with essential thrombocythemia (ET) and 5.3% of cases with primary myelofibrosis (PMF). Moreover, 25% of polycythemia vera, 29.5% of ET, and 48.1% of PMF were negative for all three mutations (JAK2V617F, MPL, and CALR). Compared with those patients with JAK2V617F mutation, CALR-mutated ET patients displayed unique hematological phenotypes, including higher platelet counts, and lower leukocyte counts and hemoglobin levels. Significant differences were not found between Chinese PMF patients with mutants CALR and JAK2V617F in terms of laboratory features. Interestingly, patients with CALR mutations showed markedly decreased levels of leukocyte alkaline phosphatase (LAP) expression, whereas those with JAK2V617F mutation presented with elevated levels. Overall, a lower mutant rate of CALR gene and a higher triple-negative rate were identified in the cohort of Chinese patients with MPNs. This result indicates that an undiscovered mutant gene may have a significant role in these patients. Moreover, these pathological features further imply that the disease biology varies considerably between mutants CALR and JAK2V617F. PMID:26375990

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2016-08-01

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

  14. Microscopic and mesoscopic structural features of an activated carbon sample, prepared from sorghum via activation by phosphoric acid

    SciTech Connect

    Temleitner, László; Pusztai, László; Rubio-Arroyo, Manuel F.; Aguilar-López, Sergio; Pizio, Orest

    2012-12-15

    Graphical abstract: Display Omitted Highlights: ► Preparation of a new activated carbon sample from sorghum. ► Characterization by adsorption/desorption methods. ► Determination of the structure by synchrotron X-ray diffraction. ► The sample is amorphous and contains distorted graphene fragments. ► A characteristic nanoscale distance is established from the radial distribution function. -- Abstract: An acidic chemical activation procedure has been used for preparing activated carbon with a surface area exceeding 1000 m{sup 2}/g from sorghum. In order to reveal structural features, synchrotron X-ray diffraction measurements have been performed. The structure of the material has been characterized by the total scattering structure factor and the radial distribution function describing short-range arrangement of atoms at distances of the order of a few atomic diameters as well as correlations at a longer scale, of the order of nanometers. The atomic arrangement has been found to be consistent with that of amorphous graphite-like carbon. As far as the mesoscopic structure is concerned, the presence of a characteristic distance is suggested on the basis of the clear nanometer scale oscillations of the radial distribution function, which distance may be assigned as the mesopore size in the material. It is suggested that the approach devized here may later be applied routinely for other activated carbon samples, too, for characterizing atomic and nanoscale order simultaneously.

  15. Unsupervised Feature Learning Improves Prediction of Human Brain Activity in Response to Natural Images

    PubMed Central

    Güçlü, Umut; van Gerven, Marcel A. J.

    2014-01-01

    Encoding and decoding in functional magnetic resonance imaging has recently emerged as an area of research to noninvasively characterize the relationship between stimulus features and human brain activity. To overcome the challenge of formalizing what stimulus features should modulate single voxel responses, we introduce a general approach for making directly testable predictions of single voxel responses to statistically adapted representations of ecologically valid stimuli. These representations are learned from unlabeled data without supervision. Our approach is validated using a parsimonious computational model of (i) how early visual cortical representations are adapted to statistical regularities in natural images and (ii) how populations of these representations are pooled by single voxels. This computational model is used to predict single voxel responses to natural images and identify natural images from stimulus-evoked multiple voxel responses. We show that statistically adapted low-level sparse and invariant representations of natural images better span the space of early visual cortical representations and can be more effectively exploited in stimulus identification than hand-designed Gabor wavelets. Our results demonstrate the potential of our approach to better probe unknown cortical representations. PMID:25101625

  16. Features of the electronic structure of the active center of an HbS molecule

    NASA Astrophysics Data System (ADS)

    Novoselov, D. Yu.; Korotin, Dm. M.; Anisimov, V. I.

    2016-01-01

    Features of the electronic structure of the nonprotein part of the mutant form of the human hemoglobin molecule, HbS, are studied along with the magnetic state of the iron ion that is the "nucleus" of the active center of the molecule. It is found that the mutant form of the HbS molecule differs from a normal hemoglobin molecule by the distortion of the local environment of the iron ion, which changes the energy level splitting by a crystal field. As a result of ab initio calculations, the magnetic transition in the iron atom from the high-spin state to the low-spin state upon the addition of molecular oxygen to hemoglobin molecule is reproduced. It is established for the first time that a change in the crystal and electronic structure of the active center as a result of a mutation can lead to a substantial change in the energy of the bond between the active center of the hemoglobin molecule and an oxygen molecule.

  17. Medicinal properties of mangiferin, structural features, derivative synthesis, pharmacokinetics and biological activities.

    PubMed

    Benard, Outhiriaradjou; Chi, Yuling

    2015-01-01

    The identification of biologically active and potentially therapeutically useful pharmacophores from natural products has been a long-term focus in the pharmaceutical industry. The recent emergence of a worldwide obesity and Type II diabetes epidemic has increased focus upon small molecules that can modulate energy metabolism, insulin sensitivity and fat biology. Interesting preliminary work done on mangiferin (MGF), the predominant constituent of extracts of the mango plant Mangifera indica L., portends potential for this pharmacophore as a novel parent compound for treating metabolic disorders. MGF is comprised of a C-glucosylated xanthone. Owing to the xanthone chemical structure, MGF has a redox active aromatic system and has antioxidant properties. MGF exerts varied and impressive metabolic effects in animals, improving metabolic disorders. For example we have discovered that MGF is a novel activator of the mammalian pyruvate dehydrogenase complex, leading to enhancement of carbohydrate utilization in oxidative metabolism, and leading to increased insulin sensitivity in animal models of obesity and insulin resistance. In addition, recent unbiased proteomics studies revealed that MGF upregulates proteins pivotal for mitochondrial bioenergetics and downregulates proteins controlling de novo lipogenesis in liver, helping to explain protective effects of MGF in prevention of liver steatosis. Several chemical studies have achieved synthesis of MGF, suggesting possible synthetic strategies to alter its chemical structure for development of structure-activity relationship (SAR) information. Ultimately, chemical derivatization studies could lead to the eventual development of novel therapeutics based upon the parent pharmacophore structure. Here we provide comprehensive review on chemical features of MGF, synthesis of its derivatives, its pharmacokinetics and biological activities. PMID:25827900

  18. Image sensor innovations for low light levels with active imaging features

    NASA Astrophysics Data System (ADS)

    Powell, Gareth H.; Fereyre, Pierre

    2015-02-01

    Advances in CMOS imaging enable image capture at lower light levels. Color detection is also possible where human vision becomes less sensitive in night conditions. In daytime conditions, there are a number of climatic conditions such as rain, fog, snow or smoke etc. that render traditional `intelligent' outdoor cameras that perform various forms of detection and identification tasks relatively ineffective. It has been proven that an adapted five transistor pixel CMOS sensor can perform range-gated active imaging that extends considerably the usability of intelligent cameras in the most difficult conditions. This paper discusses advanced state of the art image sensors with embedded features, with emphasis on the everimportant size, weight, power and cost benefits and discusses the new applications that are enabled.

  19. Feature Selection for Wearable Smartphone-Based Human Activity Recognition with Able bodied, Elderly, and Stroke Patients

    PubMed Central

    2015-01-01

    Human activity recognition (HAR), using wearable sensors, is a growing area with the potential to provide valuable information on patient mobility to rehabilitation specialists. Smartphones with accelerometer and gyroscope sensors are a convenient, minimally invasive, and low cost approach for mobility monitoring. HAR systems typically pre-process raw signals, segment the signals, and then extract features to be used in a classifier. Feature selection is a crucial step in the process to reduce potentially large data dimensionality and provide viable parameters to enable activity classification. Most HAR systems are customized to an individual research group, including a unique data set, classes, algorithms, and signal features. These data sets are obtained predominantly from able-bodied participants. In this paper, smartphone accelerometer and gyroscope sensor data were collected from populations that can benefit from human activity recognition: able-bodied, elderly, and stroke patients. Data from a consecutive sequence of 41 mobility tasks (18 different tasks) were collected for a total of 44 participants. Seventy-six signal features were calculated and subsets of these features were selected using three filter-based, classifier-independent, feature selection methods (Relief-F, Correlation-based Feature Selection, Fast Correlation Based Filter). The feature subsets were then evaluated using three generic classifiers (Naïve Bayes, Support Vector Machine, j48 Decision Tree). Common features were identified for all three populations, although the stroke population subset had some differences from both able-bodied and elderly sets. Evaluation with the three classifiers showed that the feature subsets produced similar or better accuracies than classification with the entire feature set. Therefore, since these feature subsets are classifier-independent, they should be useful for developing and improving HAR systems across and within populations. PMID:25885272

  20. Feature selection for wearable smartphone-based human activity recognition with able bodied, elderly, and stroke patients.

    PubMed

    Capela, Nicole A; Lemaire, Edward D; Baddour, Natalie

    2015-01-01

    Human activity recognition (HAR), using wearable sensors, is a growing area with the potential to provide valuable information on patient mobility to rehabilitation specialists. Smartphones with accelerometer and gyroscope sensors are a convenient, minimally invasive, and low cost approach for mobility monitoring. HAR systems typically pre-process raw signals, segment the signals, and then extract features to be used in a classifier. Feature selection is a crucial step in the process to reduce potentially large data dimensionality and provide viable parameters to enable activity classification. Most HAR systems are customized to an individual research group, including a unique data set, classes, algorithms, and signal features. These data sets are obtained predominantly from able-bodied participants. In this paper, smartphone accelerometer and gyroscope sensor data were collected from populations that can benefit from human activity recognition: able-bodied, elderly, and stroke patients. Data from a consecutive sequence of 41 mobility tasks (18 different tasks) were collected for a total of 44 participants. Seventy-six signal features were calculated and subsets of these features were selected using three filter-based, classifier-independent, feature selection methods (Relief-F, Correlation-based Feature Selection, Fast Correlation Based Filter). The feature subsets were then evaluated using three generic classifiers (Naïve Bayes, Support Vector Machine, j48 Decision Tree). Common features were identified for all three populations, although the stroke population subset had some differences from both able-bodied and elderly sets. Evaluation with the three classifiers showed that the feature subsets produced similar or better accuracies than classification with the entire feature set. Therefore, since these feature subsets are classifier-independent, they should be useful for developing and improving HAR systems across and within populations. PMID:25885272

  1. Probing structural features of Alzheimer's amyloid-β pores in bilayers using site-specific amino acid substitutions.

    PubMed

    Capone, Ricardo; Jang, Hyunbum; Kotler, Samuel A; Kagan, Bruce L; Nussinov, Ruth; Lal, Ratnesh

    2012-01-24

    A current hypothesis for the pathology of Alzheimer's disease (AD) proposes that amyloid-β (Aβ) peptides induce uncontrolled, neurotoxic ion flux across cellular membranes. The mechanism of ion flux is not fully understood because no experiment-based Aβ channel structures at atomic resolution are currently available (only a few polymorphic states have been predicted by computational models). Structural models and experimental evidence lend support to the view that the Aβ channel is an assembly of loosely associated mobile β-sheet subunits. Here, using planar lipid bilayers and molecular dynamics (MD) simulations, we show that amino acid substitutions can be used to infer which residues are essential for channel structure. We created two Aβ(1-42) peptides with point mutations: F19P and F20C. The substitution of Phe19 with Pro inhibited channel conductance. MD simulation suggests a collapsed pore of F19P channels at the lower bilayer leaflet. The kinks at the Pro residues in the pore-lining β-strands induce blockage of the solvated pore by the N-termini of the chains. The cysteine mutant is capable of forming channels, and the conductance behavior of F20C channels is similar to that of the wild type. Overall, the mutational analysis of the channel activity performed in this work tests the proposition that the channels consist of a β-sheet rich organization, with the charged/polar central strand containing the mutation sites lining the pore, and the C-terminal strands facing the hydrophobic lipid tails. A detailed understanding of channel formation and its structure should aid studies of drug design aiming to control unregulated Aβ-dependent ion fluxes. PMID:22242635

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

    PubMed Central

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

    2014-01-01

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

  3. Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites*

    PubMed Central

    Haerteis, Silke; Krappitz, Annabel; Krappitz, Matteus; Murphy, Jane E.; Bertog, Marko; Krueger, Bettina; Nacken, Regina; Chung, Hyunjae; Hollenberg, Morley D.; Knecht, Wolfgang; Bunnett, Nigel W.; Korbmacher, Christoph

    2014-01-01

    Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK178), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases. PMID:24841206

  4. Active site models for the Cu(A) site of peptidylglycine α-hydroxylating monooxygenase and dopamine β-monooxygenase.

    PubMed

    Kunishita, Atsushi; Ertem, Mehmed Z; Okubo, Yuri; Tano, Tetsuro; Sugimoto, Hideki; Ohkubo, Kei; Fujieda, Nobutaka; Fukuzumi, Shunichi; Cramer, Christopher J; Itoh, Shinobu

    2012-09-01

    A mononuclear copper(II) superoxo species has been invoked as the key reactive intermediate in aliphatic substrate hydroxylation by copper monooxygenases such as peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM). We have recently developed a mononuclear copper(II) end-on superoxo complex using a N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane tridentate ligand, the structure of which is similar to the four-coordinate distorted tetrahedral geometry of the copper-dioxygen adduct found in the oxy-form of PHM (Prigge, S. T.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Science2004, 304, 864-867). In this study, structures and physicochemical properties as well as reactivity of the copper(I) and copper(II) complexes supported by a series of tridentate ligands having the same N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane framework have been examined in detail to shed light on the chemistry dictated in the active sites of mononuclear copper monooxygenases. The ligand exhibits unique feature to stabilize the copper(I) complexes in a T-shape geometry and the copper(II) complexes in a distorted tetrahedral geometry. Low temperature oxygenation of the copper(I) complexes generated the mononuclear copper(II) end-on superoxo complexes, the structure and spin state of which have been further characterized by density functional theory (DFT) calculations. Detailed kinetic analysis on the O(2)-adduct formation reaction gave the kinetic and thermodynamic parameters providing mechanistic insights into the association and dissociation processes of O(2) to the copper complexes. The copper(II) end-on superoxo complex thus generated gradually decomposed to induce aliphatic ligand hydroxylation. Kinetic and DFT studies on the decomposition reaction have suggested that C-H bond abstraction occurs unimolecularly from the superoxo complex with subsequent rebound of the copper hydroperoxo species to generate the oxygenated

  5. Active site conformational changes of prostasin provide a new mechanism of protease regulation by divalent cations

    SciTech Connect

    Spraggon, Glen; Hornsby, Michael; Shipway, Aaron; Tully, David C.; Bursulaya, Badry; Danahay, Henry; Harris, Jennifer L.; Lesley, Scott A.

    2010-01-12

    Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca{sup +2}. Comparison of the structures with each other and with other members of the trypsin-like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca{sup +2} cations. This induced fit active site provides a new possible mode of regulation of trypsin-like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.

  6. Warrego Valles and Other Candidate Sites of Local Hydrothermal Activity Within The Thaumasia Region, Mars

    NASA Technical Reports Server (NTRS)

    Dohm, J. M.; Tanaka, K. L.; Lias, J. H.; Hare, T. M.; Anderson, R. C.; Gulick, V. C.

    1998-01-01

    We have previously demonstrated for the Thaumasia region of Mars that: (1) valley formation peaked during the Noachian and declined substantially during the Hesperian and Amazonian Periods and (2) valleys, many of which form networking systems, largely occur near volcanoes, highly faulted terrains, and large impact craters of similar age, thus suggesting hydrothermal activity. In Tanaka et al, the various hypotheses for valley formation on Mars are presented, and a geologic explanation for valley erosion in the Thaumasia region is given that "best fits" the region's geographic and geologic datasets. That comprehensive GIS-based investigation suggests that hydrothermal and seismic activity were the primary causes of valley formation in the Thaumasia region; the data make widespread precipitation less likely as a major factor in valley formation, except perhaps during the Early Noachian, for which much of the geologic record has been destroyed. Based on the reconstruction of the stratigraphic, tectonic, volcanic, and erosional histories and the close association of valleys in time and space with Noachian to Early Hesperian volcanoes and rift systems and Hesperian to Early Amazonian impact craters less than 50 km in diameter, we propose 13 sites of hydrothermal activity within the Thaumasia region; these are the best examples of valleys associated with these geologic features, but there are other less pronounced correlations elsewhere in the region.

  7. Conversed mutagenesis of an inactive peptide to ASIC3 inhibitor for active sites determination.

    PubMed

    Osmakov, Dmitry I; Koshelev, Sergey G; Andreev, Yaroslav A; Dyachenko, Igor A; Bondarenko, Dmitry A; Murashev, Arkadii N; Grishin, Eugene V; Kozlov, Sergey A

    2016-06-15

    Peptide Ugr9-1 from the venom of sea anemone Urticina grebelnyi selectively inhibits the ASIC3 channel and significantly reverses inflammatory and acid-induced pain in vivo. A close homolog peptide Ugr 9-2 does not have these features. To find the pharmacophore residues and explore structure-activity relationships of Ugr 9-1, we performed site-directed mutagenesis of Ugr 9-2 and replaced several positions by the corresponding residues from Ugr 9-1. Mutant peptides Ugr 9-2 T9F and Ugr 9-2 Y12H were able to inhibit currents of the ASIC3 channels 2.2 times and 1.3 times weaker than Ugr 9-1, respectively. Detailed analysis of the spatial models of Ugr 9-1, Ugr 9-2 and both mutant peptides revealed the presence of the basic-aromatic clusters on opposite sides of the molecule, each of which is responsible for the activity. Additionally, Ugr9-1 mutant with truncated N- and C-termini retained similar with the Ugr9-1 action in vitro and was equally potent in vivo model of thermal hypersensitivity. All together, these results are important for studying the structure-activity relationships of ligand-receptor interaction and for the future development of peptide drugs from animal toxins. PMID:26686983

  8. Combining EEG Microstates with fMRI Structural Features for Modeling Brain Activity.

    PubMed

    Michalopoulos, Kostas; Bourbakis, Nikolaos

    2015-12-01

    Combining information from Electroencephalography (EEG) and Functional Magnetic Resonance Imaging (fMRI) has been a topic of increased interest recently. The main advantage of the EEG is its high temporal resolution, in the scale of milliseconds, while the main advantage of fMRI is the detection of functional activity with good spatial resolution. The advantages of each modality seem to complement each other, providing better insight in the neuronal activity of the brain. The main goal of combining information from both modalities is to increase the spatial and the temporal localization of the underlying neuronal activity captured by each modality. This paper presents a novel technique based on the combination of these two modalities (EEG, fMRI) that allow a better representation and understanding of brain activities in time. EEG is modeled as a sequence of topographies, based on the notion of microstates. Hidden Markov Models (HMMs) were used to model the temporal evolution of the topography of the average Event Related Potential (ERP). For each model the Fisher score of the sequence is calculated by taking the gradient of the trained model parameters. The Fisher score describes how this sequence deviates from the learned HMM. Canonical Partial Least Squares (CPLS) were used to decompose the two datasets and fuse the EEG and fMRI features. In order to test the effectiveness of this method, the results of this methodology were compared with the results of CPLS using the average ERP signal of a single channel. The presented methodology was able to derive components that co-vary between EEG and fMRI and present significant differences between the two tasks. PMID:26584584

  9. Using Citizen Scientists to Gather, Analyze, and Disseminate Information About Neighborhood Features That Affect Active Living.

    PubMed

    Winter, Sandra J; Goldman Rosas, Lisa; Padilla Romero, Priscilla; Sheats, Jylana L; Buman, Matthew P; Baker, Cathleen; King, Abby C

    2016-10-01

    Many Latinos are insufficiently active, partly due to neighborhoods with little environmental support for physical activity. Multi-level approaches are needed to create health-promoting neighborhoods in disadvantaged communities. Participant "citizen scientists" were adolescent (n = 10, mean age = 12.8 ± 0.6 years) and older adult (n = 10, mean age = 71.3 ± 6.5 years), low income Latinos in North Fair Oaks, California. Citizen scientists conducted environmental assessments to document perceived barriers to active living using the Stanford Healthy Neighborhood Discovery Tool, which records GPS-tracked walking routes, photographs, audio narratives, and survey responses. Using a community-engaged approach, citizen scientists subsequently attended a community meeting to engage in advocacy training, review assessment data, prioritize issues to address and brainstorm potential solutions and partners. Citizen scientists each conducted a neighborhood environmental assessment and recorded 366 photographs and audio narratives. Adolescents (n = 4), older adults (n = 7) and community members (n = 4) collectively identified reducing trash and improving personal safety and sidewalk quality as the priority issues to address. Three adolescent and four older adult citizen scientists volunteered to present study findings to key stakeholders. This study demonstrated that with minimal training, low-income, Latino adolescent and older adult citizen scientists can: (1) use innovative technology to gather information about features of their neighborhood environment that influence active living, (2) analyze their information and identify potential solutions, and (3) engage with stakeholders to advocate for the development of healthier neighborhoods. PMID:26184398

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

    PubMed Central

    Stec, Boguslaw

    2012-01-01

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

  11. 78 FR 33908 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-05

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

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

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-03

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

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

    DOE Data Explorer

    John Peterson

    2015-04-17

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

  14. Premature skin aging features rescued by inhibition of NADPH oxidase activity in XPC-deficient mice.

    PubMed

    Hosseini, Mohsen; Mahfouf, Walid; Serrano-Sanchez, Martin; Raad, Houssam; Harfouche, Ghida; Bonneu, Marc; Claverol, Stephane; Mazurier, Frederic; Rossignol, Rodrigue; Taieb, Alain; Rezvani, Hamid Reza

    2015-04-01

    Xeroderma pigmentosum type C (XP-C) is characterized mostly by a predisposition to skin cancers and accelerated photoaging, but little is known about premature skin aging in this disease. By comparing young and old mice, we found that the level of progerin and p16(INK4a) expression, β-galactosidase activity, and reactive oxygen species, which increase with age, were higher in young Xpc(-/-) mice than in young Xpc(+/+) ones. The expression level of mitochondrial complexes and mitochondrial functions in the skin of young Xpc(-/-) was as low as in control aged Xpc(+/+)animals. Furthermore, the metabolic profile in young Xpc(-/-) mice resembled that found in aged Xpc(+/+) mice. Furthermore, premature skin aging features in young Xpc(-/-) mice were mostly rescued by inhibition of nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) activity by using a NOX1 peptide inhibitor, suggesting that the continuous oxidative stress due to overactivation of NOX1 has a causative role in the underlying pathophysiology. PMID:25437426

  15. Nuclear Site Security in the Event of Terrorist Activity

    SciTech Connect

    Thomson, M.L.; Sims, J.

    2008-07-01

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

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

    SciTech Connect

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

    1994-09-01

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

  17. Strong Nonadditivity as a Key Structure–Activity Relationship Feature: Distinguishing Structural Changes from Assay Artifacts

    PubMed Central

    2015-01-01

    Nonadditivity in protein–ligand affinity data represents highly instructive structure–activity relationship (SAR) features that indicate structural changes and have the potential to guide rational drug design. At the same time, nonadditivity is a challenge for both basic SAR analysis as well as many ligand-based data analysis techniques such as Free-Wilson Analysis and Matched Molecular Pair analysis, since linear substituent contribution models inherently assume additivity and thus do not work in such cases. While structural causes for nonadditivity have been analyzed anecdotally, no systematic approaches to interpret and use nonadditivity prospectively have been developed yet. In this contribution, we lay the statistical framework for systematic analysis of nonadditivity in a SAR series. First, we develop a general metric to quantify nonadditivity. Then, we demonstrate the non-negligible impact of experimental uncertainty that creates apparent nonadditivity, and we introduce techniques to handle experimental uncertainty. Finally, we analyze public SAR data sets for strong nonadditivity and use recourse to the original publications and available X-ray structures to find structural explanations for the nonadditivity observed. We find that all cases of strong nonadditivity (ΔΔpKi and ΔΔpIC50 > 2.0 log units) with sufficient structural information to generate reasonable hypothesis involve changes in binding mode. With the appropriate statistical basis, nonadditivity analysis offers a variety of new attempts for various areas in computer-aided drug design, including the validation of scoring functions and free energy perturbation approaches, binding pocket classification, and novel features in SAR analysis tools. PMID:25760829

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

    SciTech Connect

    Miao, Yinglong; Baudry, Jerome Y

    2011-01-01

    Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.

  19. The luminous polycyclic aromatic hydrocarbon emission features: Applications to high redshift galaxies and active galactic nuclei

    NASA Astrophysics Data System (ADS)

    Shipley, Heath Vernon

    (corrected for attenuation using the mono-chromatic rest-frame 24um emission), with a tight scatter of 0.15 dex. The scatter is comparable to that between SFRs derived from the Paalpha and dust-corrected Halpha emission lines, implying the PAH features may be as accurate a SFR indicator as the Hydrogen recombination lines. Because the PAH features are so bright, our PAH SFR calibration enables an efficient way to measure SFRs in distant galaxies with JWST to SFRs as low as ~10 M; yr-1 to z >~ 2. We use Spitzer/IRS observations of PAH features in lensed star-forming galaxies at 1 < z < 3 to demonstrate the utility of the PAHs to derive SFRs as accurate as those available from Paalpha. Chapter 4 is the application of the PAH SFRs for galaxies with AGN to demonstrate the reliability for studies of the co-evolution of star-formation and SMBH accretion. We present a study of the contribution from star-formation in galaxies of varying AGN activity (from pure star-forming galaxies to quasars) as a function of total IR luminosity using a sample of 220 galaxies. We use mid-IR spectroscopy from the Spitzer/IRS and photometry from the MIPS mum, 70mum and 160mum bands with partial coverage of the sample with the Herschel 160mum band for the quasars. The contribution from star-formation to the total IR luminosity implied by the PAH emission decreases with increasing IR luminosity. We find a similar result to previous studies for the correlation between SFR, i.e. PAH luminosity, and AGN luminosity for quasars of LSF [special characters omitted] for the 11.3mum PAH feature only (which has been shown to be the most reliable PAH feature in the vicinity of AGN). This may indicate the PAH luminosity remains a reliable tracer of the SFR for galaxies with strong AGN contributions (i.e. quasars), as we did not subtract off the AGN component before measuring the SFR from the PAH luminosity.

  20. The active site of hen egg-white lysozyme: flexibility and chemical bonding

    SciTech Connect

    Held, Jeanette Smaalen, Sander van

    2014-04-01

    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. Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts.

    PubMed

    Wang, Lu-Cun; Friend, C M; Fushimi, Rebecca; Madix, Robert J

    2016-07-01

    The activation of molecular O2 as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O2 activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O2 dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O2 dissociation is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O2 dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction. PMID:27376884

  2. Active Site Structure and Peroxidase Activity of Oxidatively Modified Cytochrome c Species in Complexes with Cardiolipin.

    PubMed

    Capdevila, Daiana A; Oviedo Rouco, Santiago; Tomasina, Florencia; Tortora, Verónica; Demicheli, Verónica; Radi, Rafael; Murgida, Daniel H

    2015-12-29

    We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to those of the wild-type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high-spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the measured apparent binding affinities for CL are ∼4 times larger than for wild-type Cyt-c. On the basis of these results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein. PMID:26620444

  3. Activation and maintenance of peripheral semantic features of unambiguous words after right hemisphere brain damage in adults

    PubMed Central

    Tompkins, Connie A.; Fassbinder, Wiltrud; Scharp, Victoria L.; Meigh, Kimberly M.

    2009-01-01

    Background The right cerebral hemisphere (RH) sustains activation of subordinate, secondary, less common, and/or distantly related meanings of words. Much of the pertinent data come from studies of homonyms, but some evidence also suggests that the RH has a unique maintenance function in relation to unambiguous nouns. In a divided visual field priming study, Atchley, Burgess, and Keeney (1999) reported that only left visual field/RH presentation yielded evidence of continuing activation of peripheral semantic features that were incompatible with the most common image or representation of their corresponding nouns (e.g., rotten for “apple”). Activation for weakly related features that were compatible with the dominant representation (e.g., crunchy) was sustained over time regardless of the visual field/hemisphere of initial stimulus input. Several studies report that unilateral right hemisphere brain damage (RHD) in adults affects the RH’s meaning maintenance function, but this work also has centred on homonyms, and/or more recently metonymic and metaphoric polysemous words. Aims The current investigation examined whether RHD deficits in processing secondary and/or distantly related meanings of words, typically observed in studies of homonyms, would extend to peripheral, weakly related semantic features of unambiguous nouns. Methods & Procedures Participants were 28 adults with unilateral RHD from cerebrovascular accident, and 38 adults without brain damage. Participants listened to spoken sentences that ended with an unambiguous noun. Each sentence was followed by a spoken target phoneme string. Targets included peripheral semantic features of the sentence-final noun that were either compatible or incompatible with the dominant mental images of the noun, and were presented at two intervals after that noun. A lexical decision task was used to gauge both the early activation and maintenance of activation for these weakly related semantic features. Outcomes

  4. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles

    PubMed Central

    2015-01-01

    Mineral dusts originating from Earth’s crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts. Here we investigated in closer detail the reasons for its activity and the difference in the activity of the different feldspars. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. K-feldspar showed by far the highest ice nucleation activity. Finally, we give a potential explanation of this effect, finding alkali-metal ions having different hydration shells and thus an influence on the ice nucleation activity of feldspar surfaces. PMID:25584435

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

    SciTech Connect

    Not Available

    1993-01-27

    On October 24, 1992, President Bush signed into law the National Energy Policy Act of 1992. The bill is a sweeping, comprehensive overhaul of the Nation`s energy laws, the first in more than a decade. Among other provisions, the National Energy Policy Act reforms the licensing process for new nuclear power plants by adopting a new approach developed by the US Nuclear Regulatory Commission (NRC) in 1989, and upheld in court in 1992. The NRC 10 CFR Part 52 rule is a three-step process that guarantees public participation at each step. The steps are: early site permit approval; standard design certifications; and, combined construction/operating licenses for nuclear power reactors. Licensing reform increases an organization`s ability to respond to future baseload electricity generation needs with less financial risk for ratepayers and the organization. Costly delays can be avoided because design, safety and siting issues will be resolved before a company starts to build a plant. Specifically, early site permit approval allows for site suitability and acceptability issues to be addressed prior to an organization`s commitment to build a plant. Responsibility for site-specific activities, including communications and public participation, rests with those organizations selected to try out early site approval. This plan has been prepared to assist those companies (referred to as sponsoring organizations) in planning their communications and public involvement programs. It provides research findings, information and recommendations to be used by organizations as a resource and starting point in developing their own plans.

  6. Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide reductase.

    PubMed

    Kapetanaki, Sofia M; Field, Sarah J; Hughes, Ross J L; Watmough, Nicholas J; Liebl, Ursula; Vos, Marten H

    2008-01-01

    The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in approximately 13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site. PMID:18420024

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-07-13

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

  9. Current Challenges for Modeling Enzyme Active Sites by Biomimetic Synthetic Diiron Complexes

    PubMed Central

    Friedle, Simone; Reisner, Erwin; Lippard, Stephen J.

    2010-01-01

    This tutorial review describes recent progress in modeling the active sites of carboxylate-rich non-heme diiron enzymes that activate dioxygen to carry out several key reactions in nature. The chemistry of soluble methane monooxygenase, which catalyzes the selective oxidation of methane to methanol, is of particular interest for (bio)technological applications. Novel synthetic diiron complexes that mimic structural, and, to a lesser extent, functional features of these diiron enzymes are discussed. The chemistry of the enzymes is also briefly summarized. A particular focus of this review is on models that mimic characteristics of the diiron systems that were previously not emphasized, including systems that contain (i) aqua ligands, (ii) different substrates tethered to the ligand framework, (iii) dendrimers attached to carboxylates to mimic the protein environment, (iv) two N-donors in a syn-orientation with respect to the iron-iron vector, and (v) a N-rich ligand environment capable of accessing oxygenated high-valent diiron intermediates. PMID:20485834

  10. Structure of Arabidopsis thaliana 5-methylthioribose Kinase Reveals a More Occluded Active Site Than its Bacterial Homolog

    SciTech Connect

    Ku,S.; Cornell, K.; Howell, P.

    2007-01-01

    Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics. The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATP?S and MTR has been determined at 1.9 Angstroms resolution. The structure is similar to B. subtilis MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of A. thaliana and B. subtilis MTR kinase adopt different conformations despite high sequence similarity. The ATP?S analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the A. thaliana enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR. The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase.

  11. Structural Characterization of Mutations at the Oxygen Activation Site in Monomeric Sarcosine Oxidase

    SciTech Connect

    Schuman Jorns, Marilyn; Chen, Zhi-wei; Mathews, F. Scott

    2010-04-30

    Oxygen reduction and sarcosine oxidation in monomeric sarcosine oxidase (MSOX) occur at separate sites above the si- and re-faces, respectively, of the flavin ring. Mutagenesis studies implicate Lys265 as the oxygen activation site. Substitution of Lys265 with a neutral (Met, Gln, or Ala) or basic (Arg) residue results in an {approx}10{sup 4}- or 250-fold decrease, respectively, in the reaction rate. The overall structure of MSOX and residue conformation in the sarcosine binding cavity are unaffected by replacement of Lys265 with Met or Arg. The side chain of Met265 exhibits the same configuration in each molecule of Lys265Met crystals and is nearly congruent with Lys265 in wild-type MSOX. The side chain of Arg265 is, however, dramatically shifted (4-5 {angstrom}) compared with Lys265, points in the opposite direction, and exhibits significant conformational variability between molecules of the same crystal. The major species in solutions of Lys265Arg is likely to contain a 'flipped-out' Arg265 and exhibit negligible oxygen activation, similar to Lys265Met. The 400-fold higher oxygen reactivity observed with Lys265Arg is attributed to a minor (<1%) 'flipped-in' Arg265 conformer whose oxygen reactivity is similar to that of wild-type MSOX. A structural water (WAT1), found above the si-face of the flavin ring in all previously determined MSOX structures, is part of an apparent proton relay system that extends from FAD N(5) to bulk solvent. WAT1 is strikingly absent in Lys265Met and Lys265Arg, a feature that may account for the apparent kinetic stabilization of a reductive half-reaction intermediate that is detectable with the mutants but not wild-type MSOX.

  12. Synthetic Active Site Model of the [NiFeSe] Hydrogenase

    PubMed Central

    Wombwell, Claire; Reisner, Erwin

    2015-01-01

    A dinuclear synthetic model of the [NiFeSe] hydrogenase active site and a structural, spectroscopic and electrochemical analysis of this complex is reported. [NiFe(‘S2Se2’)(CO)3] (H2‘S2Se2’=1,2-bis(2-thiabutyl-3,3-dimethyl-4-selenol)benzene) has been synthesized by reacting the nickel selenolate complex [Ni(‘S2Se2’)] with [Fe(CO)3bda] (bda=benzylideneacetone). X-ray crystal structure analysis confirms that [NiFe(‘S2Se2’)(CO)3] mimics the key structural features of the enzyme active site, including a doubly bridged heterobimetallic nickel and iron center with a selenolate terminally coordinated to the nickel center. Comparison of [NiFe(‘S2Se2’)(CO)3] with the previously reported thiolate analogue [NiFe(‘S4’)(CO)3] (H2‘S4’=H2xbsms=1,2-bis(4-mercapto-3,3-dimethyl-2-thiabutyl)benzene) showed that the selenolate groups in [NiFe(‘S2Se2’)(CO)3] give lower carbonyl stretching frequencies in the IR spectrum. Electrochemical studies of [NiFe(‘S2Se2’)(CO)3] and [NiFe(‘S4’)(CO)3] demonstrated that both complexes do not operate as homogenous H2 evolution catalysts, but are precursors to a solid deposit on an electrode surface for H2 evolution catalysis in organic and aqueous solution. PMID:25847470

  13. Revealing divergent evolution, identifying circular permutations and detecting active-sites by protein structure comparison

    PubMed Central

    Chen, Luonan; Wu, Ling-Yun; Wang, Yong; Zhang, Shihua; Zhang, Xiang-Sun

    2006-01-01

    Background Protein structure comparison is one of the most important problems in computational biology and plays a key role in protein structure prediction, fold family classification, motif finding, phylogenetic tree reconstruction and protein docking. Results We propose a novel method to compare the protein structures in an accurate and efficient manner. Such a method can be used to not only reveal divergent evolution, but also identify circular permutations and further detect active-sites. Specifically, we define the structure alignment as a multi-objective optimization problem, i.e., maximizing the number of aligned atoms and minimizing their root mean square distance. By controlling a single distance-related parameter, theoretically we can obtain a variety of optimal alignments corresponding to different optimal matching patterns, i.e., from a large matching portion to a small matching portion. The number of variables in our algorithm increases with the number of atoms of protein pairs in almost a linear manner. In addition to solid theoretical background, numerical experiments demonstrated significant improvement of our approach over the existing methods in terms of quality and efficiency. In particular, we show that divergent evolution, circular permutations and active-sites (or structural motifs) can be identified by our method. The software SAMO is available upon request from the authors, or from and . Conclusion A novel formulation is proposed to accurately align protein structures in the framework of multi-objective optimization, based on a sequence order-independent strategy. A fast and accurate algorithm based on the bipartite matching algorithm is developed by exploiting the special features. Convergence of computation is shown in experiments and is also theoretically proven. PMID:16948858

  14. Mutations in LRP5 cause primary osteoporosis without features of OI by reducing Wnt signaling activity

    PubMed Central

    2012-01-01

    Background Primary osteoporosis is a rare childhood-onset skeletal condition whose pathogenesis has been largely unknown. We have previously shown that primary osteoporosis can be caused by heterozygous missense mutations in the Low-density lipoprotein receptor-related protein 5 (LRP5) gene, and the role of LRP5 is further investigated here. Methods LRP5 was analyzed in 18 otherwise healthy children and adolescents who had evidence of osteoporosis (manifested as reduced bone mineral density i.e. BMD, recurrent peripheral fractures and/or vertebral compression fractures) but who lacked the clinical features of osteogenesis imperfecta (OI) or other known syndromes linked to low BMD. Also 51 controls were analyzed. Methods used in the genetic analyses included direct sequencing and multiplex ligation-dependent probe amplification (MLPA). In vitro studies were performed using luciferase assay and quantitative real-time polymerase chain reaction (qPCR) to examine the effect of two novel and three previously identified mutations on the activity of canonical Wnt signaling and on expression of tryptophan hydroxylase 1 (Tph1) and 5-hydroxytryptamine (5-Htr1b). Results Two novel LRP5 mutations (c.3446 T > A; p.L1149Q and c.3553 G > A; p.G1185R) were identified in two patients and their affected family members. In vitro analyses showed that one of these novel mutations together with two previously reported mutations (p.C913fs, p.R1036Q) significantly reduced the activity of the canonical Wnt signaling pathway. Such reductions may lead to decreased bone formation, and could explain the bone phenotype. Gut-derived Lrp5 has been shown to regulate serotonin synthesis by controlling the production of serotonin rate-limiting enzyme, Tph1. LRP5 mutations did not affect Tph1 expression, and only one mutant (p.L1149Q) reduced expression of serotonin receptor 5-Htr1b (p < 0.002). Conclusions Our results provide additional information on the role of LRP5 mutations and their effects on

  15. Features of the planetary distribution of ion precipitation at different levels of magnetic activity

    NASA Astrophysics Data System (ADS)

    Vorobjev, V. G.; Yagodkina, O. I.; Antonova, E. E.

    2015-09-01

    Observations from DMSP F6 and F7 spacecraft were used to examine the features of the planetary distribution of ion precipitation. Ion characteristics were defined within the boundaries of different types of auroral electron precipitation, which in accordance with the conclusions from (Starkov et al., 2002) were divided into a structured precipitation of an auroral oval (AOP) and zones of diffuse precipitation DAZ and SDP located equatorward and poleward of AOP, respectively. Analogous to electron precipitation, ion precipitation did not demonstrate dependences of the average energy and the average energy flux of precipitating particles on the Dst index value. In the diffuse precipitation zone (DAZ) equatorward of the auroral oval, ion energies clearly peaked in the sector of 1500-1800 MLT. The average energy value grows as magnetic activity increases from ~12 keV at AL =-1000 nT to ~18 keV at AL =-1000 nT. In the region of structured precipitation (AOP), the minimum of the average ion energy is observed in the dawn sector of 0600-0900 MLT. Ion energy fluxes ( F i ) are maximal in the nighttime MLT sectors. In the zone of soft diffuse precipitation (SDP) poleward of AOP, the highest ion energy fluxes are observed in the daytime sector, while the nightside F i values are insignificant. Ion energy fluxes in the SDP zone show an anticorrelation with the average ion energy in the same MLT sector. An ion precipitation model was created which yields a global distribution of both the average ion energies and the ion energy fluxes depending on the magnetic activity expressed by AL and Dst indices. Comparison of this model with the model of electron precipitation shows that the planetary power of ion precipitation at low magnetic activity (| AL| = 100 nT) is ~12% of the electron precipitation power and exponentially decreases to ~4% at | AL| > 1000 nT. The ion precipitation model was used to calculate the plasma pressure at the ionospheric altitudes. The planetary

  16. Common features and peculiarities of the seismic activity at Phlegraean Fields, Long Valley, and Vesuvius

    USGS Publications Warehouse

    Marzocchi, W.; Vilardo, G.; Hill, D.P.; Ricciardi, G.P.; Ricco, C.

    2001-01-01

    We analyzed and compared the seismic activity that has occurred in the last two to three decades in three distinct volcanic areas: Phlegraean Fields, Italy; Vesuvius, Italy; and Long Valley, California. Our main goal is to identify and discuss common features and peculiarities in the temporal evolution of earthquake sequences that may reflect similarities and differences in the generating processes between these volcanic systems. In particular, we tried to characterize the time series of the number of events and of the seismic energy release in terms of stochastic, deterministic, and chaotic components. The time sequences from each area consist of thousands of earthquakes that allow a detailed quantitative analysis and comparison. The results obtained showed no evidence for either deterministic or chaotic components in the earthquake sequences in Long Valley caldera, which appears to be dominated by stochastic behavior. In contrast, earthquake sequences at Phlegrean Fields and Mount Vesuvius show a deterministic signal mainly consisting of a 24-hour periodicity. Our analysis suggests that the modulation in seismicity is in some way related to thermal diurnal processes, rather than luni-solar tidal effects. Independently from the process that generates these periodicities on the seismicity., it is suggested that the lack (or presence) of diurnal cycles is seismic swarms of volcanic areas could be closely linked to the presence (or lack) of magma motion.

  17. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

    PubMed Central

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W.; Gruber, Karl; Macheroux, Peter

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family. PMID:27276217

  18. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts.

    PubMed

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and (57)Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  19. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    NASA Astrophysics Data System (ADS)

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-10-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity.

  20. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    PubMed Central

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  1. Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

    HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites. PMID:26786892

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

    SciTech Connect

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

    2008-11-10

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

  3. Utility of passive photography to objectively audit built environment features of active transport journeys: an observational study

    PubMed Central

    2013-01-01

    Background Active transport can contribute to physical activity accumulation and improved health in adults. The built environment is an established associate of active transport behaviours; however, assessment of environmental features encountered during journeys remains challenging. The purpose of this study was to examine the utility of wearable cameras to objectively audit and quantify environmental features along work-related walking and cycling routes. Methods A convenience sample of employed adults was recruited in New Zealand, in June 2011. Participants wore a SenseCam for all journeys over three weekdays and completed travel diaries and demographic questionnaires. SenseCam images for work-related active transport journeys were coded for presence of environmental features hypothesised to be related to active transport. Differences in presence of features by transport mode and in participant-reported and SenseCam-derived journey duration were determined using two-sample tests of proportion and an independent samples t-test, respectively. Results Fifteen adults participated in the study, yielding 1749 SenseCam images from 30 work-related active transport journeys for coding. Significant differences in presence of features were found between walking and cycling journeys. Almost a quarter of images were uncodeable due to being too dark to determine features. There was a non-significant tendency for respondents to under-report their journey duration. Conclusion This study provides proof of concept for the use of the SenseCam to capture built environment data in real time that may be related to active transportation. Further work is required to test and refine coding methodologies across a range of settings, travel behaviours, and demographic groups. PMID:23575288

  4. Highly Selective Adsorption of Ethylene over Ethane in a MOF Featuring the Combination of Open Metal Site and -Complexation

    DOE PAGESBeta

    Zhang, Yiming; Li, Baiyan; Wu, Zili; Ma, Shengqian

    2015-01-09

    The introduction of the combination of open metal site (OMS) and -complexation into MOF has led to very high ethylene/ethane adsorption selectivity at 318K, as illustrated in the context of MIL-101-Cr-SO3Ag. The interactions with ethylene from both OMS and -complexation in MIL-101-Cr-SO3Ag have been investigated by in situ IR spectroscopic studies and computational calculations, which suggest -complexation contributes dominantly to the high ethylene/ethane adsorption selectivity.

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

    PubMed

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes. PMID:25449264

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

    SciTech Connect

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G.

    2014-01-03

    equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.

  7. Feature Statistics Modulate the Activation of Meaning during Spoken Word Processing

    ERIC Educational Resources Information Center

    Devereux, Barry J.; Taylor, Kirsten I.; Randall, Billi; Geertzen, Jeroen; Tyler, Lorraine K.

    2016-01-01

    Understanding spoken words involves a rapid mapping from speech to conceptual representations. One distributed feature-based conceptual account assumes that the statistical characteristics of concepts' features--the number of concepts they occur in ("distinctiveness/sharedness") and likelihood of co-occurrence ("correlational…

  8. Characterization of an Active Thermal Erosion Site, Caribou Creek, Alaska

    NASA Astrophysics Data System (ADS)

    Busey, R.; Bolton, W. R.; Cherry, J. E.; Hinzman, L. D.

    2013-12-01

    The goal of this project is to estimate volume loss of soil over time from this site, provide parameterizations on erodibility of ice rich permafrost and serve as a baseline for future landscape evolution simulations. Located in the zone of discontinuous permafrost, the interior region of Alaska (USA) is home to a large quantity of warm, unstable permafrost that is both high in ice content and has soil temperatures near the freezing point. Much of this permafrost maintains a frozen state despite the general warming air temperature trend in the region due to the presence of a thick insulating organic mat and a dense root network in the upper sub-surface of the soil column. At a rapidly evolving thermo-erosion site, located within the Caribou-Poker Creeks Research Watershed (part of the Bonanza Creek LTER) near Chatanika, Alaska (N65.140, W147.570), the protective organic layer and associated plants were disturbed by an adjacent traditional use trail and the shifting of a groundwater spring. These triggers have led to rapid geomorphological change on the landscape as the soil thaws and sediment is transported into the creek at the valley bottom. Since 2006 (approximately the time of initiation), the thermal erosion has grown to 170 meters length, 3 meters max depth, and 15 meters maximum width. This research combines several data sets: DGPS survey, imagery from an extremely low altitude pole-based remote sensing (3 to 5 meters above ground level), and imagery from an Unmanned Aerial System (UAS) at about 60m altitude.

  9. Marine Biology Field Trip Sites. Ocean Related Curriculum Activities.

    ERIC Educational Resources Information Center

    Pauls, John

    The ocean affects all of our lives. Therefore, awareness of and information about the interconnections between humans and oceans are prerequisites to making sound decisions for the future. Project ORCA (Ocean Related Curriculum Activities) has developed interdisciplinary curriculum materials designed to meet the needs of students and teachers…

  10. Autobiographical Planning and the Brain: Activation and Its Modulation by Qualitative Features.

    PubMed

    Spreng, R Nathan; Gerlach, Kathy D; Turner, Gary R; Schacter, Daniel L

    2015-11-01

    To engage in purposeful behavior, it is important to make plans, which organize subsequent actions. Most studies of planning involve "look-ahead" puzzle tasks that are unrelated to personal goals. We developed a task to assess autobiographical planning, which involves the formulation of personal plans in response to real-world goals, and examined autobiographical planning in 63 adults during fMRI scanning. Autobiographical planning was found to engage the default network, including medial-temporal lobe and midline structures, and executive control regions in lateral pFC and parietal cortex and caudate. To examine how specific qualitative features of autobiographical plans modulate neural activity, we performed parametric modulation analyses. Ratings of plan detail, novelty, temporal distance, ease of plan formulation, difficulty in goal completion, and confidence in goal accomplishment were used as covariates in six hierarchical linear regression models. This modeling procedure removed shared variance among the ratings, allowing us to determine the independent relationship between ratings of interest and trial-wise BOLD signal. We found that specific autobiographical planning, describing a detailed, achievable, and actionable planning process for attaining a clearly envisioned future, recruited both default and frontoparietal brain regions. In contrast, abstract autobiographical planning, plans that were constructed from more generalized semantic or affective representations of a less tangible and distant future, involved interactions among default, sensory perceptual, and limbic brain structures. Specific qualities of autobiographical plans are important predictors of default and frontoparietal control network engagement during plan formation and reflect the contribution of mnemonic and executive control processes to autobiographical planning. PMID:26102226

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

    PubMed

    Macomber, Lee; Minkara, Mona S; Hausinger, Robert P; Merz, Kenneth M

    2015-02-23

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes, and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  12. Reduction of Urease Activity by Interaction with the Flap Covering the Active Site

    PubMed Central

    Macomber, Lee; Minkara, Mona S.; Hausinger, Robert P.; Merz, Kenneth M.

    2015-01-01

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  13. Encroachment of Human Activity on Sea Turtle Nesting Sites

    NASA Astrophysics Data System (ADS)

    Ziskin, D.; Aubrecht, C.; Elvidge, C.; Tuttle, B.; Baugh, K.; Ghosh, T.

    2008-12-01

    The encroachment of anthropogenic lighting on sea turtle nesting sites poses a serious threat to the survival of these animals [Nicholas, 2001]. This danger is quantified by combining two established data sets. The first is the Nighttime Lights data produced by the NOAA National Geophysical Data Center [Elvidge et al., 1997]. The second is the Marine Turtle Database produced by the World Conservation Monitoring Centre (WCMC). The technique used to quantify the threat of encroachment is an adaptation of the method described in Aubrecht et al. [2008], which analyzes the stress on coral reef systems by proximity to nighttime lights near the shore. Nighttime lights near beaches have both a direct impact on turtle reproductive success since they disorient hatchlings when they mistake land-based lights for the sky-lit surf [Lorne and Salmon, 2007] and the lights are also a proxy for other anthropogenic threats. The identification of turtle nesting sites with high rates of encroachment will hopefully steer conservation efforts to mitigate their effects [Witherington, 1999]. Aubrecht, C, CD Elvidge, T Longcore, C Rich, J Safran, A Strong, M Eakin, KE Baugh, BT Tuttle, AT Howard, EH Erwin, 2008, A global inventory of coral reef stressors based on satellite observed nighttime lights, Geocarto International, London, England: Taylor and Francis. In press. Elvidge, CD, KE Baugh, EA Kihn, HW Kroehl, ER Davis, 1997, Mapping City Lights with Nighttime Data from the DMSP Operational Linescan System, Photogrammatic Engineering and Remote Sensing, 63:6, pp. 727-734. Lorne, JK, M Salmon, 2007, Effects of exposure to artificial lighting on orientation of hatchling sea turtles on the beach and in the ocean, Endangered Species Research, Vol. 3: 23-30. Nicholas, M, 2001, Light Pollution and Marine Turtle Hatchlings: The Straw that Breaks the Camel's Back?, George Wright Forum, 18:4, p77-82. Witherington, BE, 1999, Reducing Threats To Nesting Habitat, Research and Management Techniques for

  14. Small Molecule Active Site Directed Tools for Studying Human Caspases.

    PubMed

    Poreba, Marcin; Szalek, Aleksandra; Kasperkiewicz, Paulina; Rut, Wioletta; Salvesen, Guy S; Drag, Marcin

    2015-11-25

    Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases, or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes. PMID:26551511

  15. Activation of brown adipose tissue mitochondrial GDP binding sites

    SciTech Connect

    Swick, A.G.

    1987-01-01

    The primary function of brown adipose tissue (BAT) is heat production. This ability is attributed to the existence of a unique inner mitochondrial membrane protein termed the uncoupling protein or thermogenin. This protein is permeable to H+ and thus allows respiration (and therefore thermogenesis) to proceed at a rapid rate, independent of ADP phosphorylation. Proton conductance can be inhibited by the binding of purine nucleotides to the uncoupling protein. The binding of (/sup 3/H)-GDP to BAT mitochondria is frequently used as a measure of BAT thermogenic activity. Rats fed a diet that was low but adequate in protein exhibited a decrease in feed efficiency. In addition, BAT thermogenesis was activated as indicated by an elevation in the level of GDP binding to BAT mitochondria. This phenomena occurred in older rats and persisted over time.

  16. Highly Selective Adsorption of Ethylene over Ethane in a MOF Featuring the Combination of Open Metal Site and -Complexation

    SciTech Connect

    Zhang, Yiming; Li, Baiyan; Wu, Zili; Ma, Shengqian

    2015-01-09

    The introduction of the combination of open metal site (OMS) and -complexation into MOF has led to very high ethylene/ethane adsorption selectivity at 318K, as illustrated in the context of MIL-101-Cr-SO3Ag. The interactions with ethylene from both OMS and -complexation in MIL-101-Cr-SO3Ag have been investigated by in situ IR spectroscopic studies and computational calculations, which suggest -complexation contributes dominantly to the high ethylene/ethane adsorption selectivity.

  17. Monoaminergic tone supports conductance correlations and stabilizes activity features in pattern generating neurons of the lobster, Panulirus interruptus.

    PubMed

    Krenz, Wulf-Dieter; Parker, Anna R; Rodgers, Edmund; Baro, Deborah J

    2015-01-01

    Experimental and computational studies demonstrate that different sets of intrinsic and synaptic conductances can give rise to equivalent activity patterns. This is because the balance of conductances, not their absolute values, defines a given activity feature. Activity-dependent feedback mechanisms maintain neuronal conductance correlations and their corresponding activity features. This study demonstrates that tonic nM concentrations of monoamines enable slow, activity-dependent processes that can maintain a correlation between the transient potassium current (I(A) and the hyperpolarization activated current (Ih) over the long-term (i.e., regulatory change persists for hours after removal of modulator). Tonic 5 nM DA acted through an RNA interference silencing complex (RISC)- and RNA polymerase II-dependent mechanism to maintain a long-term positive correlation between I(A) and Ih in the lateral pyloric neuron (LP) but not in the pyloric dilator neuron (PD). In contrast, tonic 5 nM 5HT maintained a RISC-dependent positive correlation between I(A) and Ih in PD but not LP over the long-term. Tonic 5 nM OCT maintained a long-term negative correlation between I(A) and Ih in PD but not LP; however, it was only revealed when RISC was inhibited. This study also demonstrated that monoaminergic tone can also preserve activity features over the long-term: the timing of LP activity, LP duty cycle and LP spike number per burst were maintained by tonic 5 nM DA. The data suggest that low-level monoaminergic tone acts through multiple slow processes to permit cell-specific, activity-dependent regulation of ionic conductances to maintain conductance correlations and their corresponding activity features over the long-term. PMID:26539083

  18. Monoaminergic tone supports conductance correlations and stabilizes activity features in pattern generating neurons of the lobster, Panulirus interruptus

    PubMed Central

    Krenz, Wulf-Dieter; Parker, Anna R.; Rodgers, Edmund; Baro, Deborah J.

    2015-01-01

    Experimental and computational studies demonstrate that different sets of intrinsic and synaptic conductances can give rise to equivalent activity patterns. This is because the balance of conductances, not their absolute values, defines a given activity feature. Activity-dependent feedback mechanisms maintain neuronal conductance correlations and their corresponding activity features. This study demonstrates that tonic nM concentrations of monoamines enable slow, activity-dependent processes that can maintain a correlation between the transient potassium current (IA) and the hyperpolarization activated current (Ih) over the long-term (i.e., regulatory change persists for hours after removal of modulator). Tonic 5 nM DA acted through an RNA interference silencing complex (RISC)- and RNA polymerase II-dependent mechanism to maintain a long-term positive correlation between IA and Ih in the lateral pyloric neuron (LP) but not in the pyloric dilator neuron (PD). In contrast, tonic 5 nM 5HT maintained a RISC-dependent positive correlation between IA and Ih in PD but not LP over the long-term. Tonic 5 nM OCT maintained a long-term negative correlation between IA and Ih in PD but not LP; however, it was only revealed when RISC was inhibited. This study also demonstrated that monoaminergic tone can also preserve activity features over the long-term: the timing of LP activity, LP duty cycle and LP spike number per burst were maintained by tonic 5 nM DA. The data suggest that low-level monoaminergic tone acts through multiple slow processes to permit cell-specific, activity-dependent regulation of ionic conductances to maintain conductance correlations and their corresponding activity features over the long-term. PMID:26539083

  19. Digital reconstruction on geographical environment of Neolithic human activities in the Lingjiatan site of Chaohu City, East China

    NASA Astrophysics Data System (ADS)

    Wang, Xinyuan; Zhang, Jie; Wu, Li; Zhou, Kunshu; Mo, Duowen

    2010-11-01

    The Chaohu Lake Basin is an important area for ancient human activities in East China. The Lingjiatan site, which is located at the southeast of Chaohu City, Anhui Province, and 35 km north to the Yangtze River and 5 km south to the Taihu Mountain, is the most representative Neolithic Age site with advanced jade-carving techniques in this area. The 14C date of Lingjiatan Site is about 5600~5300aBP, the same time as the Hongshan culture and earlier than the Liangzhu culture, which falls into the Mid-Holocene epoch. Based on mid-high resolution remote sensing images and former archaeological materials, combined with field investigations and sampling analysis of the archaeological site profile of Lingjiatan Site as well as core drillings in the Chaohu Lake, the paper reconstructs the climate environment of the Lingjiatan site and the environmental background of ancient human activities during Mid-Holocene. The research results show that: (1) The ancients in Lingjiatan lived in the Holocene Optimum, its culture development was during the interim phase when the climate transformed from warm and wet to cool and dry. (2) The ground surface deposited in the last phase of late Pleistocene epoch (OSL dating is 11.6 +/-1.0 ka BP) was the living ground for Lingjiatan ancient humans. The sedimentary discontinuous surface may be caused by strong fluvial erosion under the warm and humid climatic conditions of the Mid-Holocene. (3) Originally, paleo-geomorphic surface was a level shallow mesa foreside southern part of Taihu Mountain, but was cut by fluvial waters and the geomorphologic configuration formed "finger-like" features alternately with strip hillocks and rivers. These features can be seen on the Landsat ETM+ remote sensing image, especially the depression area. This depression is now cropland, and was interpreted as the palaeochannels. (4) Based on the remote sensing image interpretation, the site was in a "peninsula shape" environment which had rivers flowing around the

  20. Digital reconstruction on geographical environment of Neolithic human activities in the Lingjiatan site of Chaohu City, East China

    NASA Astrophysics Data System (ADS)

    Wang, Xinyuan; Zhang, Jie; Wu, Li; Zhou, Kunshu; Mo, Duowen

    2009-09-01

    The Chaohu Lake Basin is an important area for ancient human activities in East China. The Lingjiatan site, which is located at the southeast of Chaohu City, Anhui Province, and 35 km north to the Yangtze River and 5 km south to the Taihu Mountain, is the most representative Neolithic Age site with advanced jade-carving techniques in this area. The 14C date of Lingjiatan Site is about 5600~5300aBP, the same time as the Hongshan culture and earlier than the Liangzhu culture, which falls into the Mid-Holocene epoch. Based on mid-high resolution remote sensing images and former archaeological materials, combined with field investigations and sampling analysis of the archaeological site profile of Lingjiatan Site as well as core drillings in the Chaohu Lake, the paper reconstructs the climate environment of the Lingjiatan site and the environmental background of ancient human activities during Mid-Holocene. The research results show that: (1) The ancients in Lingjiatan lived in the Holocene Optimum, its culture development was during the interim phase when the climate transformed from warm and wet to cool and dry. (2) The ground surface deposited in the last phase of late Pleistocene epoch (OSL dating is 11.6 +/-1.0 ka BP) was the living ground for Lingjiatan ancient humans. The sedimentary discontinuous surface may be caused by strong fluvial erosion under the warm and humid climatic conditions of the Mid-Holocene. (3) Originally, paleo-geomorphic surface was a level shallow mesa foreside southern part of Taihu Mountain, but was cut by fluvial waters and the geomorphologic configuration formed "finger-like" features alternately with strip hillocks and rivers. These features can be seen on the Landsat ETM+ remote sensing image, especially the depression area. This depression is now cropland, and was interpreted as the palaeochannels. (4) Based on the remote sensing image interpretation, the site was in a "peninsula shape" environment which had rivers flowing around the

  1. N2O reduction by the mu4-sulfide-bridged tetranuclear CuZ cluster active site.

    PubMed

    Chen, Peng; Gorelsky, Serge I; Ghosh, Somdatta; Solomon, Edward I

    2004-08-13

    Nitrous oxide (N2O) reduction is a chemical challenge both in the selective oxidation of organic substrates by N2O and in the removal of N2O as a green-house gas. The reduction of N2O is thermodynamically favorable but kinetically inert, and requires activating transition-metal centers. In biological systems, N2O reduction is the last step in the denitrification process of the bacterial nitrogen cycle and is accomplished by the enzyme nitrous oxide reductase, whose active site consists of a micro4-sulfide-bridged tetranuclear CuZ cluster which has many unusual spectroscopic features. Recent studies have developed a detailed electronic-structure description of the resting CuZ cluster, determined its catalytically relevant state, and provided insight into the role of this tetranuclear copper cluster in N2O activation and reduction. PMID:15307074

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

    PubMed

    Muramatsu, Akiyoshi

    2016-01-01

    The "School Health and Safety Act" was enforced in April 2009 in Japan, and "school environmental health standards" were established by the Minister of Education, Culture, Sports, Science and Technology. In Article 24 of the Enforcement Regulations, the duties of the school pharmacist have been clarified; school pharmacists have charged with promoting health activities in schools and carrying out complete and regular checks based on the "school environmental health standards" in order to protect the health of students and staff. In supported of this, the school pharmacist group of Japan Pharmaceutical Association has created and distributed digital video discs (DVDs) on "check methods of school environmental health standards" as support material. We use the DVD to ensure the basic issues that school pharmacists deal with, such as objectives, criteria, and methods for each item to be checked, advice, and post-measures. We conduct various workshops and classes, and set up Q&A committees so that inquiries from members are answered with the help of such activities. In addition, school pharmacists try to improve the knowledge of the school staff on environmental hygiene during their in-service training. They also conduct "drug abuse prevention classes" at school and seek to improve knowledge and recognition of drugs, including "dangerous drugs". PMID:27252053

  3. Kinetic evidence for an anion binding pocket in the active site of nitronate monooxygenase.

    PubMed

    Francis, Kevin; Gadda, Giovanni

    2009-10-01

    A series of monovalent, inorganic anions and aliphatic aldehydes were tested as inhibitors for Hansenula mrakii and Neurospora crassa nitronate monooxygenase, formerly known as 2-nitropropane dioxygenase, to investigate the structural features that contribute to the binding of the anionic nitronate substrates to the enzymes. A linear correlation between the volumes of the inorganic anions and their effectiveness as competitive inhibitors of the enzymes was observed in a plot of pK(is)versus the ionic volume of the anion with slopes of 0.041+/-0.001 mM/A(3) and 0.027+/-0.001 mM/A(3) for the H. mrakii and N. crassa enzymes, respectively. Aliphatic aldehydes were weak competitive inhibitors of the enzymes, with inhibition constants that are independent of their alkyl chain lengths. The reductive half reactions of H. mrakii nitronate monooxygenase with primary nitronates containing two to four carbon atoms all showed apparent K(d) values of approximately 5 mM. These results are consistent with the presence of an anion binding pocket in the active site of nitronate monooxygenase that interacts with the nitro group of the substrate, and suggest a minimal contribution of the hydrocarbon chain of the nitronates to the binding of the ligands to the enzyme. PMID:19683782

  4. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors*

    PubMed Central

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

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

    PubMed

    Matsubu, John C; Yang, Vanessa N; Christopher, Phillip

    2015-03-01

    CO2 reduction by H2 on heterogeneous catalysts is an important class of reactions that has been studied for decades. However, atomic scale details of structure-function relationships are still poorly understood. Particularly, it has been suggested that metal particle size plays a unique role in controlling the stability of CO2 hydrogenation catalysts and the distribution of active sites, which dictates reactivity and selectivity. These studies often have not considered the possible role of isolated metal active sites in the observed dependences. Here, we utilize probe molecule diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) with known site-specific extinction coefficients to quantify the fraction of Rh sites residing as atomically dispersed isolated sites (Rhiso), as well as Rh sites on the surface of Rh nanoparticles (RhNP) for a series of TiO2 supported Rh catalysts. Strong correlations were observed between the catalytic reverse water gas shift turn over frequency (TOF) and the fraction of Rhiso sites and between catalytic methanation TOF and the fraction of RhNP sites. Furthermore, it was observed that reaction condition-induced disintegration of Rh nanoparticles, forming Rhiso active sites, controls the changing reactivity with time on stream. This work demonstrates that isolated atoms and nanoparticles of the same metal on the same support can exhibit uniquely different catalytic selectivity in competing parallel reaction pathways and that disintegration of nanoparticles under reaction conditions can play a significant role in controlling stability. PMID:25671686

  6. Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

    PubMed Central

    Lerch, Michael T.; Horwitz, Joseph; McCoy, John; Hubbell, Wayne L.

    2013-01-01

    Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875–85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate. PMID:24248390

  7. Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin.

    PubMed

    Lerch, Michael T; Horwitz, Joseph; McCoy, John; Hubbell, Wayne L

    2013-12-01

    Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875-85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate. PMID:24248390

  8. Clinicopathological features, vitamin D serological levels and prognosis in cutaneous melanoma of shield-sites: an update.

    PubMed

    Paolino, Giovanni; Moliterni, Elisa; Didona, Dario; Garelli, Valentina; Corsetti, Paola; Lopez, Teresa; Richetta, Antonio Giovanni; Cantisani, Carmen; Bottoni, Ugo; Calvieri, Stefano

    2015-01-01

    Intermittent sun exposure and sunburns are strongly related to the development of melanoma (MM); however, MM can also arise in non-sun exposed areas, where other biological pathways may cause the disease, with different outcomes. At the same time, evidences of serum levels of vitamin D in melanoma patients according to sun-exposed or not-sun-exposed areas are still lacking, especially if compared with the percentage of BRAF mutation. We performed a retrospective analysis with patients registered in our electronic database and an observational study in patients with a recent diagnosis of MM. Performing Kaplan-Meier product and log-rank test, median disease-free survival was 78 months in non-shield-sites (NST-MM) patients and 20.5 months in shield-sites (ST-MM) patients (p < 0.0001); also in the long term, a better behavior was observed for NST-MM (80 vs. 42 months; p < 0.0001). Among 87 melanoma patients with a recent history of MM (≤30 days), we found that ST-MM patients showed lower values of vitamin D compared with NST-MM patients. Regarding BRAF status, a BRAF mutation was present in 13 % of ST-MM and in 41 % of NST-MM. Performing Mc-Nemar test, we found a statistical significant correlation between low serum levels of vitamin D in ST-MM and low percentage of BRAF mutation (p = 0.03), as well as between serum levels of vitamin D and high percentage of BRAF mutation in NST-MM (p < 0.001). All these aspects confirm that in ST-MM, other pathways play pivotal points, if compared with NST-MM. PMID:25516505

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

    NASA Astrophysics Data System (ADS)

    Qi, Jian-Xun; Jiang, Fan

    2011-05-01

    The crystallographic temperature factors (B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule. Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents. It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme. To test the theory of conformational flexibility of enzyme active site, crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions. It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M. It was also found that the B factors averaged over individually collected data sets were more accurate. Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets. Furthermore, we found that the correctly predicted active sites included not only the more flexible residues, but also some more rigid residues. Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network, covering the majority of the substrate binding residues. Therefore, this experimental prediction method may be useful for characterizing the binding site and the function of a protein, such as drug targeting.

  10. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  11. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  12. Extending the Diffuse Layer Model of Surface Acidity Behavior: III. Estimating Bound Site Activity Coefficients

    EPA Science Inventory

    Although detailed thermodynamic analyses of the 2-pK diffuse layer surface complexation model generally specify bound site activity coefficients for the purpose of accounting for those non-ideal excess free energies contributing to bound site electrochemical potentials, in applic...

  13. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    SciTech Connect

    Not Available

    1994-02-01

    This report is a detailed listing of all of the Hazardous Waste activities occurring at Martin Marietta`s K-25 site. Contained herein are hazardous waste notification forms, waste stream reports, generator fee forms and various TSDR reports.

  14. Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site.

    PubMed

    Zhu, Wen-Jing; Li, Miao; Wang, Xiao-Yun

    2007-12-01

    Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (DeltaH degrees ) was 12.38kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK. PMID:17765964

  15. 78 FR 8190 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-05

    ...BOEM is reopening the comment period announced in the Notice of Intent to Prepare an Environmental Assessment (EA) for Commercial Wind Leasing and Site Assessment Activities on the OCS Offshore North...

  16. Some features of the bioelectric activity of the muscles with prolonged hypokinesia

    NASA Technical Reports Server (NTRS)

    Belaya, N. A.; Amirov, R. Z.; Shaposhnikov, Y. A.; Lebedeva, I. P.; Sologub, B. S.

    1978-01-01

    The effects of prolonged hypokinesia, brought on by confinement to bed and the attendant lack of motor activity, on the bioelectric activity of muscles are studied. Electromyographic measurements of amplitude and frequency indicators of muscular bioelectric activity were analyzed.

  17. Study on elemental features of Longquan celadon at Fengdongyan kiln site in Yuan and Ming Dynasties by EDXRF

    NASA Astrophysics Data System (ADS)

    Li, L.; Feng, S. L.; Feng, X. Q.; Xu, Q.; Yan, L. T.; Ma, B.; Liu, L.

    2012-12-01

    The energy dispersive X-ray fluorescence (EDXRF) is used to determine the chemical compositions of Longquan celadon body and glaze at Fengdongyan kiln in Yuan and Ming Dynasties. In order to analyze the elemental features in different cultural periods, 196 typical Longquan celadon shards are selected including the Early Yuan (EY), the Middle and Late Yuan (MLY), the Early Ming (EM) and the Middle Ming (MM) periods. The results indicate that the contents of K2O, CaO, TiO2 and Fe2O3 for celadon body and glaze are various in different cultural periods. The b values b=RO/(RO+RO), RO represents for alkali-earth metal elements and R2O stands for alkali metal elements) show that the glazes of Longquan celadon in Yuan and Ming Dynasties belong to the category of calcium-alkali glaze. The principal component analysis (PCA) exhibits that the raw materials used for body and glaze in four periods have the inheritance relation. The only difference is that the raw materials for glaze in MM period were similar to those in EM period. The producing and firing technology of Longquan celadon had reached a higher level in the Early Ming Dynasty.

  18. Building Model NASA Satellites: Elementary Students Studying Science Using a NASA-Themed Transmedia Book Featuring Digital Fabrication Activities

    ERIC Educational Resources Information Center

    Tillman, Daniel; An, Song; Boren, Rachel; Slykhuis, David

    2014-01-01

    This study assessed the impact of nine lessons incorporating a NASA-themed transmedia book featuring digital fabrication activities on 5th-grade students (n = 29) recognized as advanced in mathematics based on their academic record. Data collected included a pretest and posttest of science content questions taken from released Virginia Standards…

  19. 47 CFR 79.110 - Complaint procedures for user interfaces, menus and guides, and activating accessibility features...

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 4 2014-10-01 2014-10-01 false Complaint procedures for user interfaces, menus and guides, and activating accessibility features on digital apparatus and navigation devices. 79.110... ACCESSIBILITY OF VIDEO PROGRAMMING Apparatus § 79.110 Complaint procedures for user interfaces, menus and...

  20. Anisotropic Covalency Contributions to Superexchange Pathways in Type One Copper Active Sites

    PubMed Central

    2015-01-01

    Type one (T1) Cu sites deliver electrons to catalytic Cu active sites: the mononuclear type two (T2) Cu site in nitrite reductases (NiRs) and the trinuclear Cu cluster in the multicopper oxidases (MCOs). The T1 Cu and the remote catalytic sites are connected via a Cys-His intramolecular electron-transfer (ET) bridge, which contains two potential ET pathways: P1 through the protein backbone and P2 through the H-bond between the Cys and the His. The high covalency of the T1 Cu–S(Cys) bond is shown here to activate the T1 Cu site for hole superexchange via occupied valence orbitals of the bridge. This covalency-activated electronic coupling (HDA) facilitates long-range ET through both pathways. These pathways can be selectively activated depending on the geometric and electronic structure of the T1 Cu site and thus the anisotropic covalency of the T1 Cu–S(Cys) bond. In NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being more efficient. Comparing the MCOs to NiRs, the second-sphere environment changes the conformation of the Cys-His pathway, which selectively activates HDA for superexchange by blue π sites for efficient turnover in catalysis. These studies show that a given protein bridge, here Cys-His, provides different superexchange pathways and electronic couplings depending on the anisotropic covalencies of the donor and acceptor metal sites. PMID:25310460

  1. The Three Mycobacterium tuberculosis Antigen 85 Isoforms Have Unique Substrates and Activities Determined by Non-active Site Regions*

    PubMed Central

    Backus, Keriann M.; Dolan, Michael A.; Barry, Conor S.; Joe, Maju; McPhie, Peter; Boshoff, Helena I. M.; Lowary, Todd L.; Davis, Benjamin G.; Barry, Clifton E.

    2014-01-01

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro216–Phe228 loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors. PMID:25028517

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

    Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

    2014-04-01

    The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins. PMID:24635441

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

    PubMed

    Schwidder, M; Santhosh Kumar, M; Brückner, A; Grünert, W

    2005-02-14

    A study of Fe-ZSM-5 catalysts with variable amounts of isolated, oligomeric and heavily aggregated Fe3+ oxo sites (as evidenced by UV-Vis and EPR spectroscopic data) and their catalytic properties in the selective catalytic reduction of NO by isobutane or by NH3 is presented, which allows development of a unified concept of the active Fe sites in these reactions, according to which isolated Fe sites catalyse both SCR reactions while oligomeric sites, though also involved in the selective reduction path, limit the catalyst performance by causing the total oxidation of the reductant. PMID:15685345

  5. Object detection and tracking with active camera on motion vectors of feature points and particle filter.

    PubMed

    Chen, Yong; Zhang, Rong-Hua; Shang, Lei; Hu, Eric

    2013-06-01

    A method based on motion vectors of feature points and particle filter has been proposed and developed for an active∕moving camera for object detection and tracking purposes. The object is detected by histogram of motion vectors first, and then, on the basis of particle filter algorithm, the weighing factors are obtained via color information. In addition, re-sampling strategy and surf feature points are used to remedy the drawback of particle degeneration. Experimental results demonstrate the practicability and accuracy of the new method and are presented in the paper. PMID:23822380

  6. Geologic framework and hydrogeologic features of the Glen Rose Limestone, Camp Bullis Training Site, Bexar County, Texas

    USGS Publications Warehouse

    Clark, Allan K.

    2003-01-01

    The Glen Rose Limestone crops out over most of the Camp Bullis Training Site in northern Bexar County, Texas, where it consists of upper and lower members and composes the upper zone and the upper part of the middle zone of the Trinity aquifer. Uncharacteristically permeable in northern Bexar County, the Glen Rose Limestone can provide avenues for recharge to and potential contamination of the downgradient Edwards aquifer, which occupies the southeastern corner of Camp Bullis. The upper member of the Glen Rose Limestone characteristically is thin-bedded and composed mostly of soft limestone and marl, and the lower Glen Rose typically is composed mostly of relatively massive, fossiliferous limestone. The upper member, about 410 to 450 feet thick at Camp Bullis, was divided in this study into five hydrogeologic subdivisions, A through E (youngest to oldest). The approximately 120-foot-thick Interval A has an abundance of caves, which is indicative of its generally well developed fracture, channel, and cavern porosity that in places provides appreciable permeability. The 120- to 150-foot-thick Interval B is similar to Interval A but with less cave development and considerably less permeability. The 10- to 20-foot-thick Interval C, a layer of partly to mostly dissolved soluble carbonate minerals, is characterized by breccia porosity, boxwork permeability, and collapse structures that typically divert ground water laterally to discharge at land surface. The 135- to 180-foot-thick Interval D generally has low porosity and little permeability with some local exceptions, most notably the caprinid biostrome just below the top of the interval, which appears to be permeable by virtue of excellent moldic, vug, fracture, and cavern porosity. The 10- to 20-foot-thick Interval E, a layer of partly to mostly dissolved evaporites similar to Interval C, has similar hydrogeologic properties and a tendency to divert ground water laterally.

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

    PubMed Central

    Gosert, R; Dollenmaier, G; Weitz, M

    1997-01-01

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

  8. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    PubMed Central

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B; Sagi, Irit; Havenith, Martina

    2012-01-01

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site. PMID:21926991

  9. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    SciTech Connect

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B.; Sagi, Irit; Havenith, Martina

    2011-09-18

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

  10. Activity Participation and Sensory Features among Children with Autism Spectrum Disorders

    ERIC Educational Resources Information Center

    Little, Lauren M.; Ausderau, Karla; Sideris, John; Baranek, Grace T.

    2015-01-01

    Sensory features are highly prevalent among children with autism spectrum disorders (ASD) and have been shown to cluster into four patterns of response, including hyperresponsiveness, hyporesponsiveness, enhanced perception, and sensory interests, repetitions and seeking behaviors. Given the lack of large-scale research on the differential effects…

  11. From Alcohol Dehydrogenase to a “One-way” Carbonyl Reductase by Active-site Redesign

    PubMed Central

    Klimacek, Mario; Nidetzky, Bernd

    2010-01-01

    Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol (Kint) was altered dramatically (104- to 105-fold) from being balanced in the wild-type enzyme (Kint ≈ 3) to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. The change in Kint reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn → Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD+-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn→Asp substitutions behaved as a slow “fructose reductase” at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8–10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively “one-way” reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis. PMID:20639204

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

    The Soil Moisture and Ocean Salinity Mission (SMOS) delivers global surface soil moisture data at high temporal resolution which is of high relevance for water management, weather and climate predictions as well as hazard analysis. In order to estimate the quality and caveats of the SMOS data at different processing levels (e.g. L1C geolocated brightness temperatures TB, L2 soil moisture SM and optical thickness TAU, L3 spatio-temporal synthesis of TB, SM and TAU), product validation in various climatic regions is a crucial issue. In the framework of the Danish Hydrological Observatory (HOBE) one such validation site has been established in the Skjern River Catchment, Denmark. The catchment is one of Europe's northernmost intensely cultivated region with environmental features related to this latitude such as very sandy soils with large organic deposits under natural vegetation and region-specific land cover such as heathland. The area is of pronounced flatness and located at a short distance to the coast line in two directions. During fall 2009, a soil moisture and soil temperature network with 30 stations has been installed to provide continuous in-situ soil moisture data feasible for upscaling and comparison with SMOS data at large scale. One SMOS pixel (44x44 km2) to be validated was chosen by maximizing its coverage of the Skjern River Catchment and minimizing the open water fraction. Prevailing environmental conditions and their respective fractions were considered for the selection of suitable network locations. To further support validation activities an airborne campaign with the passive L-band microwave radiometer EMIRAD-2, was carried out within the chosen SMOS pixel in spring 2010 to directly acquire soil moisture data at intermediate scale (few kilometers spatial resolution). Concurrent with ascending SMOS overpasses, four flights were conducted with simultaneous ground sampling of surface soil moisture and auxiliary parameters within three 2x2 km

  13. Interfacial Reaction-Driven Formation of Silica Carbonate Biomorphs with Subcellular Topographical Features and Their Biological Activity.

    PubMed

    Wang, Guocheng; Zhao, Xiaobing; Möller, Marco; Moya, Sergio E

    2015-10-28

    We report the interfacial reaction-driven formation of micro/nanostructured strontium carbonate (SrCO3) biomorphs with subcellular topographical features on strontium zinc silicate (Sr2ZnSi2O7) biomedical coatings and explore their potential use in bone tissue engineering. The resulting SrCO3 crystals build a well-integrated scaffold surface that not only prevents burst release of ions from the coating but also presents nanotopographical features similar to cellular filopodia. The surface with biomorphic crystals enhances osteoblast adhesion, upregulates the alkaline phosphatase activity, and increases collagen production, highlighting the potential of the silica carbonate biomorphs for tissue regeneration. PMID:26455427

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

    SciTech Connect

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

    2007-01-01

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

  15. Assessment of the site of ventricular activation by Fourier analysis of gated blood-pool studies

    SciTech Connect

    Links, J.M.; Raichlen, J.S.; Wagner, H.N. Jr.; Reid, P.R.

    1985-01-01

    The authors studied the use of first-harmonic Fourier analysis of gated blood-pool images to assess the site of ventricular activation in a group of 12 patients undergoing electrophysiologic pacing studies. They acquired gated blood-pool studies during pacing at up to four sites at each of two different rates. A total of 50 studies were made. At a pacing rate of 100 beats/min, when the pacing electrode was the right-ventricular outflow tract, 7/8; at the anterolateral left-ventricular wall, 4/4. When the Fourier activation site was at the right-ventricular apex, 9/9 times the pacing electrode was there; at the right-ventricular outflow tract, 7/10; in the left ventricle, 4/4. Fourier analysis of gated blood-pool studies can help identify the site of ventricular activation but is not sufficiently accurate to fully replace endocardial mapping.

  16. Acrolein Modification Impairs Key Functional Features of Rat Apolipoprotein E: Identification of Modified Sites by Mass Spectrometry

    PubMed Central

    Tran, Tuyen N.; Kosaraju, Malathi G.; Tamamizu-Kato, Shiori; Akintunde, Olayemi; Zheng, Ying; Bielicki, John K.; Pinkerton, Kent; Uchida, Koji; Lee, Yuan Yu; Narayanaswami, Vasanthy

    2014-01-01

    Apolipoprotein E (apoE), an anti-atherogenic apolipoprotein, plays a significant role in the metabolism of lipoproteins. It lowers plasma lipid levels by acting as a ligand for low-density lipoprotein receptor (LDLr) family of proteins, in addition to playing a role in promoting macrophage cholesterol efflux in atherosclerotic lesions. The objective of this study is to examine the effect of acrolein modification on the structure and function of rat apoE and to determine sites and nature of modification by mass spectrometry. Acrolein is a highly reactive aldehyde, which is generated endogenously as one of the products of lipid peroxidation and is present in the environment in pollutants such as tobacco smoke and heated oils. In initial studies, acrolein-modified apoE was identified by immunoprecipitation using an acrolein-lysine specific antibody, in the plasma of ten-week old male rats that were exposed to filtered air (FA) or low doses of environmental tobacco smoke (ETS). While both groups displayed acrolein-modified apoE in the lipoprotein fraction, the ETS group had higher levels in lipid-free fraction compared to the FA group. This observation provided the rationale to further investigate the effect of acrolein modification on rat apoE at a molecular level. Treatment of recombinant rat apoE with a 10-fold molar excess of acrolein resulted in: (i) a significant decrease in lipid-binding and cholesterol efflux abilities, (ii) impairment in the LDLr- and heparin-binding capabilities, and (iii) significant alterations in the overall stability of the protein. The disruption in the functional abilities is attributed directly or indirectly to acrolein modification yielding: an aldimine adduct at K149 and K155 (+38); a propanal adduct at K135 and K138 (+56); an Nε-(3-methylpyridinium)lysine (MP-lysine) at K64, K67 and K254 (+76), and Nε-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) derivative at position K68 (+94), as determined by Matrix-Assisted Laser

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

    SciTech Connect

    Crichlow, G.; Lubetsky, J; Leng, L; Bucala, R; Lolis, E

    2009-01-01

    Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

  18. Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form*

    PubMed Central

    Gould, Christine M.; Antal, Corina E.; Reyes, Gloria; Kunkel, Maya T.; Adams, Ryan A.; Ziyar, Ahdad; Riveros, Tania; Newton, Alexandra C.

    2011-01-01

    Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C. PMID:21715334

  19. Features and amenities of school playgrounds: A direct observation study of utilization and physical activity levels outside of school time

    PubMed Central

    2011-01-01

    Background A significant amount of research has examined whether park or playground availability is associated with physical activity. However, little research has examined whether specific features or amenities of parks or playgrounds, such as the number of unique types of playground equipment or the safety of the equipment is associated with utilization of the facility or physical activity levels while at the facility. There are no studies that use direct observation and a detailed park assessment to examine these associations. Methods Twenty urban schoolyards in the Midwest, ten of which were renovated, were included in this study. Using a detailed environmental assessment tool (i.e., Environmental Assessment of Public Recreation Spaces), information on a variety of playground attributes was collected. Using direct observation (i.e., System for Observing Play and Leisure Activity in Youth), the number of adults, girls and boys attending each schoolyard and their physical activity levels were recorded. Each schoolyard was observed ten times for 90 minutes each time outside of school hours. Clustered multivariable negative binomial regressions and linear regressions were completed to examine the association between playground attributes and utilization of the schoolyard and the proportion active on the playground, respectively. Effect modification by renovation status was also examined. Results At renovated schoolyards, the total number of play features was significantly associated with greater utilization in adults and girls; overall cleanliness was significantly associated with less utilization in girls and boys; and coverage/shade for resting features was significantly associated with greater utilization in adults and boys. At unrenovated schoolyards, overall safety was significantly associated with greater utilization in boys. No playground attribute was associated with the proportion active on the playground after adjusting for all other significant

  20. Crystal Structures and Functional Characterization of Wild Type and Active Sites Mutants of CYP101D1

    PubMed Central

    Batabyal, Dipanwita; Poulos, Thomas L.

    2014-01-01

    Although CYP101D1 and P450cam catayze the same reaction at a similar rate and share strikingly similar active site architectures, there are significance functional differences. CYP101D1 thus provides an opportunity to probe what structural and functional features must be shared and what can differ yet maintain high catalytic efficiency. Crystal structures of the cyanide complex of wild type CYP101D1 and it active site mutants, D259N and T260A, have been solved. The conformational changes in CYP101D1 upon cyanide binding are very similar to P450cam indicating a similar mechanism for proton delivery during oxygen activation using solvent assisted proton transfer. The D259N-CN− complex shows a perturbed solvent structure compared to wild type which is similar to what was observed in the oxy-complex of the corresonding D251N mutant in P450cam. As in P450cam the T260A mutant is highly uncoupled while the D259N gives barely detectable activity. Despite these similarities, CYP101D1 is able to use the P450cam redox partners while P450cam cannot use the CYP101D1 redox partners. Thus the strict requirement of P450cam for its own redox partner is relaxed in CYP101D1. Differences in the local environment of the essential Asp (Asp259 in CYP101D1) provides a strucutral basis for understanding these functional differences. PMID:24261604

  1. New Cloud Activity on Uranus in 2004: First Detection of a Southern Feature at 2.2 microns

    NASA Astrophysics Data System (ADS)

    Hammel, H. B.; de Pater, I.; Gibbard, S. G.; Lockwood, G. W.; Rages, K.

    2004-11-01

    On 4 July 2004 UT, we detected a southern hemispheric cloud feature on Uranus at K' (2.2 microns) using adaptive optics and the NIRC2 camera on the Keck 10-m. At H (1.6 microns), the feature had two components: a condensed core (2004-A) co-located with the K' feature at latitude -36.0 ± 0.4 deg, and an extended feature (2004-B) at roughly -33.7 ± 0.4 deg. When we observed again on 8 July UT, the core had faded at K'. By 9 July we were unable to detect a K' feature; the extended feature was still seen at H. The initial high K'-brightness of 2004-A indicates that the core's scattering particles reached altitudes above the 1-bar level, with the larger feature at H, 2004-B, residing below the 1.1-bar level (assuming the model of de Pater et al. 2002, Icarus 160, 359). The core's rapid disappearance at K' indicates dynamical processes in the local vertical aerosol structure, for example, subsidence of the feature's cloud tops. Features 2004-A and 2004-B had zonal velocities of 107 and 115 m/s, respectively (+/- 3 m/s), different from past velocities near these latitudes. These features are either new, or are long-lived but drifting in latitude (as has been seen on Neptune); see abstract by Rages et al. this volume. Regardless of their latitude, no southern features on Uranus have been detected at wavelengths of 2 microns or longward, indicating some change of activity. Continued observations of Uranus are strongly encouraged as its 2007 equinox approaches. HBH acknowledges partial support from NASA grants NAG5-11961 and NAG5-10451. IdP acknowledges partial support from NSF and the Technology Center for Adaptive Optics, managed by UCSC under cooperative agreement No. AST-9876783. SGG's work was performed under the auspices of the U.S. DoE, National Nuclear Security Administration by the UC, LLNL under contract No. W-7405-Eng-48.

  2. Scenarios constructed for basaltic igneous activity at Yucca Mountain and vicinity; Yucca Mountain Site Characterization Project

    SciTech Connect

    Barr, G.E.; Dunn, E.; Dockery, H.; Barnard, R.; Valentine, G.; Crowe, B.

    1993-08-01

    Basaltic volcanism has been identified as a possible future event initiating a release of radionuclides from a potential repository at the proposed Yucca Mountain high-level waste repository site. The performance assessment method set forth in the Site Characterization Plan (DOE, 1988) requires that a set of scenarios encompassing all significant radionuclide release paths to the accessible environment be described. This report attempts to catalogue the details of the interactions between the features and processes produced by basaltic volcanism in the presence of the presumed groundwater flow system and a repository structure, the engineered barrier system (EBS), and waste. This catalogue is developed in the form of scenarios. We define a scenario as a well-posed problem, starting from an initiating event or process and proceeding through a logically connected and physically possible combination or sequence of features, events, and processes (FEPs) to the release of contaminants.

  3. Understanding the effect of magnesium ion concentration on the catalytic activity of ribonuclease H through computation: Does a third metal binding site modulate endonuclease activity?

    PubMed Central

    Ho, Ming-Hsun; De Vivo, Marco; Peraro, Matteo Dal; Klein, Michael L.

    2010-01-01

    Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallographic data have located two ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, experiments have shown a dependency of the catalytic activity on the Mg2+ concentration. Moreover, in RNase H the glutamate residue E188 has been shown to be essential for full enzymatic activation regardless of the Mg2+ concentration. The catalytic center is known to contain two Mg2+ ions (Nowotny et al.) and E188 is not one of the primary metal ligands. Herein, classical molecular dynamics (MD) simulations are employed to study the metal-ligand coordination in RNase H at different concentration of Mg2+. Importantly, the presence of a third Mg2+ ion, bound to the second-shell ligand E188, is persistent feature of the MD simulations. Free energy calculations have identified two distinct conformations depending on the concentration of Mg2+. At standard concentration, a third Mg2+ is found in the catalytic pocket but it does not perturb the optimal RNase H active conformation. However, at higher concentration, the third Mg2+ ion heavily perturbs the nucleophilic water and thereby influences the catalytic efficiency of RNase H. In addition, the E188A mutant shows no ability to engage additional Mg2+ ions nearby the catalytic pocket. This finding likely explains the decrease in catalytic activity of E188A, and also supports the key role of E188 in localizing the third Mg2+ ion at the active site. Glutamate residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD

  4. Feature-specific imaging: Extensions to adaptive object recognition and active illumination based scene reconstruction

    NASA Astrophysics Data System (ADS)

    Baheti, Pawan K.

    Computational imaging (CI) systems are hybrid imagers in which the optical and post-processing sub-systems are jointly optimized to maximize the task-specific performance. In this dissertation we consider a form of CI system that measures the linear projections (i.e., features) of the scene optically, and it is commonly referred to as feature-specific imaging (FSI). Most of the previous work on FSI has been concerned with image reconstruction. Previous FSI techniques have also been non-adaptive and restricted to the use of ambient illumination. We consider two novel extensions of the FSI system in this work. We first present an adaptive feature-specific imaging (AFSI) system and consider its application to a face-recognition task. The proposed system makes use of previous measurements to adapt the projection basis at each step. We present both statistical and information-theoretic adaptation mechanisms for the AFSI system. The sequential hypothesis testing framework is used to determine the number of measurements required for achieving a specified misclassification probability. We demonstrate that AFSI system requires significantly fewer measurements than static-FSI (SFSI) and conventional imaging at low signal-to-noise ratio (SNR). We also show a trade-off, in terms of average detection time, between measurement SNR and adaptation advantage. Experimental results validating the AFSI system are presented. Next we present a FSI system based on the use of structured light. Feature measurements are obtained by projecting spatially structured illumination onto an object and collecting all of the reflected light onto a single photodetector. We refer to this system as feature-specific structured imaging (FSSI). Principal component features are used to define the illumination patterns. The optimal LMMSE operator is used to generate object estimates from the measurements. We demonstrate that this new imaging approach reduces imager complexity and provides improved image

  5. CopC protein from Pseudomonas fluorescens SBW25 features a conserved novel high-affinity Cu(II) binding site.

    PubMed

    Wijekoon, Chathuri J K; Young, Tessa R; Wedd, Anthony G; Xiao, Zhiguang

    2015-03-16

    Copper homeostasis in the bacterium Pseudomonas fluorescens SBW25 appears to be mediated mainly via chromosomal cue and cop systems. Under elevated copper levels that induce stress, the cue system is activated for expression of a P1B-type ATPase to remove excess copper from the cytosol. Under copper-limiting conditions, the cop system is activated to express two copper uptake proteins, Pf-CopCD, to import this essential nutrient. Pf-CopC is a periplasmic copper chaperone that may donate copper to the inner membrane transporter Pf-CopD for active copper importation. A database search revealed that Pf-CopC belongs to a new family of CopC proteins (designated Type B in this work) that differs significantly from the known CopC proteins of Type A that possess two separated binding sites specific for Cu(I) and Cu(II). This article reports the isolation and characterization of Pf-CopC and demonstrates that it lacks a Cu(I) binding site and possesses a novel Cu(II) site that binds Cu(II) with 100 times stronger affinity than do the Type A proteins. Presumably, this is a requirement for a copper uptake role under copper-limiting conditions. The Cu(II) site incorporates a highly conserved amino terminal copper and nickel (ATCUN) binding motif, NH2-Xxx-Xxx-His, but the anticipated ATCUN binding mode is prevented by a thermodynamically more favorable binding mode comprising His1 as a key bidentate ligand and His3 and His85 as co-ligands. However, upon His1 mutation, the ATCUN binding mode is adopted. This work demonstrates how a copper chaperone may fine tune its copper binding site to meet new challenges to its function. PMID:25710712

  6. Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents.

    PubMed

    Hudson, Elton P; Eppler, Ross K; Beaudoin, Julianne M; Dordick, Jonathan S; Reimer, Jeffrey A; Clark, Douglas S

    2009-04-01

    The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, a(w). The apparent turnover number k(cat)(app) in these systems ranged from 0.2 to 80 s(-1) and increased 11-fold in isooctane and up to 50-fold in tetrahydrofuran with increasing a(w). (19)F NMR relaxation experiments employing an active-site inhibitor were used to assess the dependence of active-site motions on a(w). The rates of NMR-derived fast (k > 10(7) s(-1)) and slow (k < 10(4) s(-1)) active-site motions increased in both solvents upon hydration, but only the slow motions correlated with k(cat). The (19)F chemical shift was a sensitive probe of the local electronic environment and provided an empirical measure of the active-site dielectric constant epsilon(as), which increased with hydration to epsilon(as) approximately 13 in each solvent. In both solvents, the transition state free energy data and epsilon(as) followed Kirkwood's model for the continuum solvation of a dipole, indicating that water also enhanced catalysis by altering the active-site's electronic environment and increasing its polarity to better stabilize the transition state. These results reveal that favorable dynamic and electrostatic effects both contribute to accelerated catalysis by solubilized subtilisin Carlsberg upon hydration in organic solvents. PMID:19317505

  7. Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site*

    PubMed Central

    Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

    2014-01-01

    Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser105 residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T5015, the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability. PMID:24448805

  8. Antiprotozoal activity of bicycles featuring a dimethylamino group at their bridgehead.

    PubMed

    Faist, Johanna; Seebacher, Werner; Saf, Robert; Brun, Reto; Kaiser, Marcel; Weis, Robert

    2016-08-15

    Several dimethylamino-derivatives of the new compound-class 3-azabicyclo[3.2.2]nonanes were prepared. For better comparison of activity also a few analogues of bicyclo[2.2.2]octanes and 2-azabicyclo[3.2.2]nonanes were synthesized. Their activities were examined in vitro against the multiresistant K1 strain of Plasmodium falciparum and against Trypanosoma brucei rhodesiense (STIB 900). A couple of the newly synthesized compounds showed promising antiprotozoal activity and selectivity. The results of the biological tests of the novel compounds were compared with the activities of already synthesized compounds and of drugs in use. Structure-activity relationships were discussed. PMID:27344215

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

    SciTech Connect

    Ashby, J.; Rowe, J.

    1980-02-01

    The primary consideration for the suitability of a nuclear waste repository site is the overall ability of the repository to safely contain radioactive waste. This report is a discussion of the past, present, and future effects of resource activities on waste containment. Past and present resource activities which provide release pathways (i.e., leaky boreholes, adjacent mines) will receive initial evaluation during the early stages of any repository site study. However, other resource activities which may have subtle effects on containment (e.g., long-term pumping causing increased groundwater gradients, invasion of saline water causing lower retardation) and all potential future resource activities must also be considered during the site evaluation process. Resource activities will affect both the siting and the designing of repositories. Ideally, sites should be located in areas of low resource activity and low potential for future activity, and repository design should seek to eliminate or minimize the adverse effects of any resource activity. Buffer zones should be created to provide areas in which resource activities that might adversely affect containment can be restricted or curtailed. This could mean removing large areas of land from resource development. The impact of these frozen assets should be assessed in terms of their economic value and of their effect upon resource reserves. This step could require a major effort in data acquisition and analysis followed by extensive numerical modeling of regional fluid flow and mass transport. Numerical models should be used to assess the effects of resource activity upon containment and should include the cumulative effects of different resource activities. Analysis by other methods is probably not possible except for relatively simple cases.

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

    PubMed

    Méthot, Nathalie; Vaillancourt, John P; Huang, JingQi; Colucci, John; Han, Yongxin; Ménard, Stéphane; Zamboni, Robert; Toulmond, Sylvie; Nicholson, Donald W; Roy, Sophie

    2004-07-01

    Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies. PMID:15067000

  11. Polarization features of solar radio emission and possible existence of current sheets in active regions

    NASA Technical Reports Server (NTRS)

    Gopalswamy, N.; Zheleznyakov, V. V.; White, S. M.; Kundu, M. R.

    1994-01-01

    We show that it is possible to account for the polarization features of solar radio emission provided the linear mode coupling theory is properly applied and the presence of current sheets in the corona is taken into account. We present a schematic model, including a current sheet that can explain the polarization features of both the low frequency slowly varying component and the bipolar noise storm radiation; the two radiations face similar propagation conditions through a current sheet and hence display similar polarization behavior. We discuss the applications of the linear mode coupling theory to the following types of solar emission: the slowly varying component, the microwave radio bursts, metric type U bursts, and bipolar noise storms.

  12. Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active site formation and catalytic specificity

    PubMed Central

    Itoh, Yuzuru; Bröcker, Markus J.; Sekine, Shun-ichi; Söll, Dieter; Yokoyama, Shigeyuki

    2015-01-01

    The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins, and is synthesized on its specific tRNA (tRNASec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNASec, formed by seryl-tRNA synthetase, to Sec-tRNASec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate (PLP)-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNASec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNASec. The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer-pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions, and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site, and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I PLP-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures. PMID:24456689

  13. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes.

    PubMed

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  14. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  15. Aerosol optical properties at a coastal site in Hong Kong, South China: temporal features, size dependencies and source analysis

    NASA Astrophysics Data System (ADS)

    Wang, Jiaping; Ding, Aijun; Virkkula, Aki; Lee, Shuncheng; Shen, Yicheng; Chi, Xuguang; Xu, Zheng

    2016-04-01

    Hong Kong is a typical coastal city adjacent to the Pearl River Delta (PRD) region in southern China, which is one of the regions suffering from severe air pollution. Atmospheric aerosols can affect the earth's radiative balance by scattering and absorbing incoming solar radiation. Black Carbon (BC) aerosol is a particularly emphasized component due to its strong light absorption. Aerosol transported from different source areas consists of distinct size distributions, leading to different optical properties. As the byproducts of the incomplete oxidation, BC and CO both have relatively long life time, their relationship is a good indicator for distinguishing different pollutant sources. In this study, temporal variations of aerosol optical properties and concentrations of BC and CO at a coastal background station in Hong Kong were investigated. Transport characteristics and origins of aerosol were elucidated by analyzing backward Lagrangian particle dispersion modeling (LPDM) results, together with related parameters including the relationships between optical properties and particle size, BC-CO correlations, ship location data and meteorological variables. From February 2012 to September 2013 and March 2014 to February 2015, continuous in-situ measurements of light scattering and absorption coefficients, particle size distribution and concentrations of BC and CO were conducted at Hok Tsui (HT), a coastal background station on the southeast tip of Hong Kong Island (22.22°N, 114.25°E, 60 m above the sea level) with few local anthropogenic activities. Affected by the Asian monsoon, this region is dominated by continental outflow in winter and by marine inflow from the South China Sea in summer, which is an ideal station for identifying the transport characteristics of aerosol and their effects on optical properties from different anthropogenic emission sources. 7-day backward Lagrangian particle dispersion modeling was performed for source identification. Three

  16. Computational approaches to the determination of active site structures and reaction mechanisms in heterogeneous catalysts.

    PubMed

    Catlow, C R A; French, S A; Sokol, A A; Thomas, J M

    2005-04-15

    We apply quantum chemical methods to the study of active site structures and reaction mechanisms in mesoporous silica and metal oxide catalysts. Our approach is based on the use of both molecular cluster and embedded cluster (QM/MM) techniques, where the active site and molecular complex are described using density functional theory (DFT) and the embedding matrix simulated by shell model potentials. We consider three case studies: alkene epoxidation over the microporous TS-1 catalyst; methanol synthesis on ZnO and Cu/ZnO and C-H bond activation over Li-doped MgO. PMID:15901543

  17. Crystal structures and functional characterization of wild-type CYP101D1 and its active site mutants.

    PubMed

    Batabyal, Dipanwita; Poulos, Thomas L

    2013-12-10

    Although CYP101D1 and P450cam catalyze the same reaction at similar rates and share strikingly similar active site architectures, there are significant functional differences. CYP101D1 thus provides an opportunity to probe what structural and functional features must be shared and what features can differ but maintain the high catalytic efficiency. Crystal structures of the cyanide complex of wild-type CYP101D1 and it active site mutants, D259N and T260A, have been determined. The conformational changes in CYP101D1 upon cyanide binding are very similar to those of P450cam, indicating a similar mechanism for proton delivery during oxygen activation using solvent-assisted proton transfer. The D259N-CN- complex shows a perturbed solvent structure compared to that of the wild type, which is similar to what was observed in the oxy complex of the corresonding D251N mutant in P450cam. As in P450cam, the T260A mutant is highly uncoupled while the D259N mutant gives barely detectable activity. Despite these similarities, CYP101D1 is able to use the P450cam redox partners while P450cam cannot use the CYP101D1 redox partners. Thus, the strict requirement of P450cam for its own redox partner is relaxed in CYP101D1. Differences in the local environment of the essential Asp (Asp259 in CYP101D1) provide a strucutral basis for understanding these functional differences. PMID:24261604

  18. Satellite SAR imagery for site discovery, change detection and monitoring activities in cultural heritage sites: experiments on the Nasca region, Peru

    NASA Astrophysics Data System (ADS)

    Tapete, D.; Cigna, F.; Masini, N.; Lasaponara, R.

    2012-04-01

    Besides their suitability for multi-temporal and spatial deformation analysis, the Synthetic Aperture Radar (SAR) image archives acquired by space-borne radar sensors can be exploited to support archaeological investigations over huge sites, even those partially or totally buried and still to be excavated. Amplitude information is one of the main properties of SAR data from which it is possible to retrieve evidences of buried structures, using feature extraction and texture analysis. Multi-temporality allows the reconstruction of past and recent evolution of both landscape and built-up environment, with the possibility to detect natural and/or anthropogenic changes, including human-induced damages to the conservation of cultural heritage. We present the methodology and first results of the experiments currently undertaken using SAR data in the Nasca region (Southern Peru), where two important civilizations such as Paracas and Nasca developed and flourished from 4th century BC to the 6th century AD. The study areas include a wide spectrum of archaeological and environmental elements to be preserved, among which: the archaeological site of Cahuachi and its surroundings, considered the largest adobe Ceremonial Centre in the World; the Nasca lines and geoglyphs in the areas of Palpa, Atarco and Nasca; the ancient networks of aqueducts and drainage galleries in the Puquios area, built by Nasca in the 1st-6th centuries AD. Archaeological prospection and multi-purpose remote sensing activities are currently carried out in the framework of the Italian mission of heritage Conservation and Archaeogeophysics (ITACA), with the direct involvement of researchers from the Institute for Archaeological and Monumental Heritage and the Institute of Methodologies for Environmental Analysis, Italian National Research Council. In this context, C- and L-band SAR images covering the Nasca region since 2001 were identified for the purposes of this research and, in particular, the following

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

    PubMed Central

    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 fluorescence results were in agreement and both paralleled enzymatic activity of papain with respect to both the variation in pH and denaturation. These results strongly suggest that SL-PMB and Acrylodan labels can be used to characterize the physical state of the active site of the enzyme. PMID:1657229

  20. Failure of origin activation in response to fork stalling leads to chromosomal instability at fragile sites.

    PubMed

    Ozeri-Galai, Efrat; Lebofsky, Ronald; Rahat, Ayelet; Bester, Assaf C; Bensimon, Aaron; Kerem, Batsheva

    2011-07-01

    Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Here, we show that even under normal growth conditions, replication fork progression along the fragile site, FRA16C, is slow and forks frequently stall at AT-rich sequences, leading to activation of additional origins to enable replication completion. Under mild replication stress, the frequency of stalling at AT-rich sequences is further increased. Strikingly, unlike in the entire genome, in the FRA16C region additional origins are not activated, suggesting that all potential origins are already activated under normal conditions. Thus, the basis for FRA16C fragility is replication fork stalling at AT-rich sequences and inability to activate additional origins under replication stress. Our results provide a mechanism explaining the replication stress sensitivity of fragile sites and thus, the basis for genomic instability during early stages of cancer development. PMID:21726815

  1. Active-site mobility revealed by the crystal structure of arylmalonate decarboxylase from Bordetella bronchiseptica.

    PubMed

    Kuettner, E Bartholomeus; Keim, Antje; Kircher, Markus; Rosmus, Susann; Sträter, Norbert

    2008-03-21

    Arylmalonate decarboxylase (AMDase) from Bordetella bronchiseptica catalyzes the enantioselective decarboxylation of arylmethylmalonates without the need for an organic cofactor or metal ion. The decarboxylation reaction is of interest for the synthesis of fine chemicals. As basis for an analysis of the catalytic mechanism of AMDase and for a rational enzyme design, we determined the X-ray structure of the enzyme up to 1.9 A resolution. Like the distantly related aspartate or glutamate racemases, AMDase has an aspartate transcarbamoylase fold consisting of two alpha/beta domains related by a pseudo dyad. However, the domain orientation of AMDase differs by about 30 degrees from that of the glutamate racemases, and also significant differences in active-site structures are observed. In the crystals, four independent subunits showing different conformations of active-site loops are present. This finding is likely to reflect the active-site mobility necessary for catalytic activity. PMID:18258259

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

    SciTech Connect

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    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.

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

    PubMed Central

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

    2015-01-01

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

  4. Coulombic effects of remote subsites on the active site of ribonuclease A.

    PubMed

    Fisher, B M; Schultz, L W; Raines, R T

    1998-12-15

    The active-site cleft of bovine pancreatic ribonuclease A (RNase A) is lined with cationic residues that interact with a bound nucleic acid. Those residues interacting with the phosphoryl groups comprise the P0, P1, and P2 subsites, with the scissile P-O5' bond residing in the P1 subsite. Coulombic interactions between the P0 and P2 subsites and phosphoryl groups of the substrate were characterized previously [Fisher, B. M., Ha, J.-H., and Raines, R. T. (1998) Biochemistry 37, 12121-12132]. Here, the interactions between these subsites and the active-site residues His12 and His119 are described in detail. A protein variant in which the cationic residues in these subsites (Lys66 in the P0 subsite and Lys7 and Arg10 in the P2 subsite) were replaced with alanine was crystallized, both free and with bound 3'-uridine monophosphate (3'-UMP). Structures of K7A/R10A/K66A RNase A and the K7A/R10A/K66A RNase A.3'-UMP complex were determined by X-ray diffraction analysis to resolutions of 2.0 and 2.1 A, respectively. There is little observable change between these structures and that of wild-type RNase A, either free or with bound 3'-cytidine monophosphate. K7A/R10A/K66A RNase A was evaluated for its ability to cleave UpA, a dinucleotide substrate that does not span the P0 or the P2 subsites. In comparison to the wild-type enzyme, the value of kcat was decreased by 5-fold and that of kcat/Km was decreased 10-fold, suggesting that these remote subsites interact with the active site. These interactions were characterized by determining the pKa values of His12 and His119 at 0.018 and 0.142 M Na+, both in wild-type RNase A and the K7A/R10A/K66A variant. The side chains of Lys7, Arg10, and Lys66 depress the pKa values of these histidine residues, and this depression is sensitive to the salt concentration. In addition, the P0 and P2 subsites influence the interaction of His12 and His119 with each other, as demonstrated by changes in the cooperativity that gives rise to microscopic

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

    PubMed

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

    1999-10-26

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

  6. Can simple interactions capture complex features of neural activity underlying behavior in a virtual reality environment?

    NASA Astrophysics Data System (ADS)

    Meshulam, Leenoy; Gauthier, Jeffrey; Brody, Carlos; Tank, David; Bialek, William

    The complex neural interactions which are abundant in most recordings of neural activity are relatively poorly understood. A prime example of such interactions can be found in the in vivo neural activity which underlies complex behaviors of mice, imaged in brain regions such as hippocampus and parietal cortex. Experimental techniques now allow us to accurately follow these neural interactions in the simultaneous activity of large neuronal populations of awake behaving animals. Here, we demonstrate that pairwise maximum entropy models can predict a surprising number of properties of the neural activity. The models, that are constrained with activity rates and interactions between pairs of neurons, are well fit to the activity `states' in the hippocampus and cortex of mice performing cognitive tasks while navigating in a virtual reality environment.

  7. Localization of the binding site of tissue-type plasminogen activator to fibrin.

    PubMed Central

    Ichinose, A; Takio, K; Fujikawa, K

    1986-01-01

    Functionally active A and B chains were separated from a two-chain form of recombinant tissue-type plasminogen activator after mild reduction and alkylation. The A chain was found to be responsible for the binding to lysine-Sepharose or fibrin and the B chain contained the catalytic activity of tissue-type plasminogen activator. An extensive reduction of two-chain tissue-type plasminogen activator, however, destroyed both the binding and catalytic activities. A thermolytic fragment, Fr. 1, of tissue-type plasminogen activator that contained a growth factor and two kringle segments retained its lysine binding activity. Additional thermolytic cleavages in the kringle-2 segment of Fr. 1 caused a total loss of the binding activity. These results indicated that the binding site of tissue-type plasminogen activator to fibrin was located in the kringle-2 segment. Images PMID:3088041

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

    SciTech Connect

    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

    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.

  9. On the importance of anandamide structural features for its interactions with DPPC bilayers: effects on PLA2 activity.

    PubMed

    Ambrosi, S; Ragni, L; Ambrosini, A; Paccamiccio, L; Mariani, P; Fiorini, R; Bertoli, E; Zolese, G

    2005-09-01

    The acylethanolamide anandamide (AEA) occurs in a variety of mammalian tissues and, as a result of its action on cannabinoid receptors, exhibits several cannabimimetic activities. Moreover, some of its effects are mediated through interaction with an ion channel-type vanilloid receptor. However, the chemical features of AEA suggest that some of its biological effects could be related to physical interactions with the lipidic part of the membrane. The present work studies the effect of AEA-induced structural modifications of the dipalmitoylphosphatidylcholine (DPPC) bilayer on phospholipase A2 (PLA2) activity, which is strictly dependent on lipid bilayer features. This study, performed by 2-dimethylamino-(6-lauroyl)-naphthalene fluorescence, demonstrates that the effect of AEA on PLA2 activity is concentration-dependent. In fact, at low AEA/DPPC molar ratios (from R = 0.001 to R = 0.04), there is an increase of the enzymatic activity, which is completely inhibited for R = 0.1. X-ray diffraction data indicate that the AEA affects DPPC membrane structural properties in a concentration-dependent manner. Because the biphasic effect of increasing AEA concentrations on PLA2 activity is related to the induced modifications of membrane bilayer structural properties, we suggest that AEA-phospholipid interactions may be important to produce, at least in part, some of the similarly biphasic responses of some physiological activities to increasing concentrations of AEA. PMID:15961786

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

    PubMed

    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

  11. Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity

    PubMed Central

    Rinehart, Jesse; Maksimova, Yelena D.; Tanis, Jessica E.; Stone, Kathryn L.; Hodson, Caleb A.; Zhang, Junhui; Risinger, Mary; Pan, Weijun; Wu, Dianqing; Colangelo, Christopher M.; Forbush, Biff; Joiner, Clinton H.; Gulcicek, Erol E.; Gallagher, Patrick G.; Lifton, Richard P.

    2010-01-01

    Summary Modulation of intracellular chloride concentration ([Cl−]i) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl− exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl−]I; however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl−]i and have implications for control of cell volume and neuronal function. PMID:19665974

  12. Active sites of ligand-protected Au25 nanoparticle catalysts for CO2 electroreduction to CO

    NASA Astrophysics Data System (ADS)

    Alfonso, Dominic R.; Kauffman, Douglas; Matranga, Christopher

    2016-05-01

    Recent experimental studies have reported the electrochemical reduction of carbon dioxide (CO2) into CO at atomically precise negatively charged Au25- nanoclusters. The studies showed CO2 conversion at remarkably low overpotentials, but the exact mechanisms and nature of the active sites remain unclear. We used first-principles density functional theory and continuum solvation models to examine the role of the cluster during electrochemical CO2 reduction and analyze the free energies of proposed intermediate species. Contrary to previous assumptions, our results show that the fully ligand protected cluster is not an active CO2 reduction catalyst because formation of the crucial carboxyl intermediate required very high electrochemical potentials. Instead, our calculations suggest that the reduction process likely occurs on a dethiolated gold site, and adsorbed carboxyl intermediate formation was significantly stabilized at dethiolated gold sites. These findings point to the crucial role of exposed metal sites during electrochemical CO2 reduction at gold nanocluster catalysts.

  13. Active sites of ligand-protected Au25 nanoparticle catalysts for CO2 electroreduction to CO.

    PubMed

    Alfonso, Dominic R; Kauffman, Douglas; Matranga, Christopher

    2016-05-14

    Recent experimental studies have reported the electrochemical reduction of carbon dioxide (CO2) into CO at atomically precise negatively charged Au25 (-) nanoclusters. The studies showed CO2 conversion at remarkably low overpotentials, but the exact mechanisms and nature of the active sites remain unclear. We used first-principles density functional theory and continuum solvation models to examine the role of the cluster during electrochemical CO2 reduction and analyze the free energies of proposed intermediate species. Contrary to previous assumptions, our results show that the fully ligand protected cluster is not an active CO2 reduction catalyst because formation of the crucial carboxyl intermediate required very high electrochemical potentials. Instead, our calculations suggest that the reduction process likely occurs on a dethiolated gold site, and adsorbed carboxyl intermediate formation was significantly stabilized at dethiolated gold sites. These findings point to the crucial role of exposed metal sites during electrochemical CO2 reduction at gold nanocluster catalysts. PMID:27179498

  14. A [Cu2O]2+ core in Cu-ZSM-5, the active site in the oxidation of methane to methanol

    PubMed Central

    Woertink, Julia S.; Smeets, Pieter J.; Groothaert, Marijke H.; Vance, Michael A.; Sels, Bert F.; Schoonheydt, Robert A.; Solomon, Edward I.

    2009-01-01

    Driven by the depletion of crude oil, the direct oxidation of methane to methanol has been of considerable interest. Promising low-temperature activity of an oxygen-activated zeolite, Cu-ZSM-5, has recently been reported in this selective oxidation and the active site in this reaction correlates with an absorption feature at 22,700 cm−1. In the present study, this absorption band is used to selectively resonance enhance Raman vibrations of this active site. 18O2 labeling experiments allow definitive assignment of the observed vibrations and exclude all previously characterized copper-oxygen species for the active site. In combination with DFT and normal coordinate analysis calculations, the oxygen activated Cu core is uniquely defined as a bent mono-(μ-oxo)dicupric site. Spectroscopically validated electronic structure calculations show polarization of the low-lying singly-occupied molecular orbital of the [Cu2O]2+ core, which is directed into the zeolite channel, upon approach of CH4. This induces significant oxyl character into the bridging O atom leading to a low transition state energy consistent with experiment and explains why the bent mono-(μ-oxo)dicupric core is highly activated for H atom abstraction from CH4. The oxygen intermediate of Cu-ZSM-5 is now the most well defined species active in the methane monooxygenase reaction. PMID:19864626

  15. Common Features of Professional Development Activities for Mathematics and Science Teachers

    ERIC Educational Resources Information Center

    Moyer-Packenham, Patricia S.; Bolyard, Johnna J.; Oh, Hana; Cerar, Nancy Irby

    2011-01-01

    This study examines professional development activities provided for mathematics and science teachers in the National Science Foundation's Math and Science Partnership Program by analyzing a cross-sectional sample of over 2000 professional development (PD) activities in the program. Data were gathered from secondary source documents and surveys to…

  16. The Luminous Polycyclic Aromatic Hydrocarbon Emission Features: Applications to High Redshift Galaxies and Active Galactic Nuclei

    NASA Astrophysics Data System (ADS)

    Shipley, Heath V.

    2016-01-01

    For decades, significant work has been applied to calibrating emission from the ultra-violet, nebular emission lines, far-infrared, X-ray and radio as tracers of the star-formation rate (SFR) in distant galaxies. Understanding the exact rate of star-formation and how it evolves with time and galaxy mass has deep implications for how galaxies form. The co-evolution of star-formation and supermassive black hole (SMBH) accretion is one of the key problems in galaxy formation theory. But, many of these SFR indicators are influenced by SMBH accretion in galaxies and result in unreliable SFRs. Utilizing the luminous polycyclic aromatic hydrocarbon (PAH) emission features, I provide a new robust SFR calibration using the luminosity emitted from the PAHs at 6.2μm, 7.7μm and 11.3μm to solve this. The PAH features emit strongly in the mid-infrared (mid-IR; 5-25μm) mitigating dust extinction, containing on average 5-10% of the total IR luminosity in galaxies. I use a sample of 105 star-forming galaxies covering a range of total IR luminosity, LIR = L(8-1000μm) = 109 - 1012 L⊙ and redshift 0 < z < 0.4, with mid-IR spectroscopy from the Spitzer Infrared Spectrograph (IRS), and data covering other SFR indicators (Hα emission and rest-frame 24μm continuum emission). The PAH luminosity correlates linearly with the SFR as measured by the Hα luminosity (corrected for attenuation using the mono-chromatic rest-frame 24μm emission), with a tight scatter of <0.15 dex. The scatter is comparable to that between SFRs derived from the Paα and dust-corrected Hα emission lines. We present a case study in advance of JWST, which will be capable of measuring SFRs (from 8μm rest-frame photometry, i.e. PAHs) in distant galaxies (z ≤ 2) with JWST/MIRI to SFRs as low as ~10 M⊙yr-1, because the PAH features are so bright. We use Spitzer/IRS observations of PAH features in lensed star-forming galaxies at 1 < z < 3 to demonstrate the utility of the PAHs to derive SFRs that agree with

  17. Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase.

    PubMed Central

    Milgrom, Y M; Murataliev, M B; Boyer, P D

    1998-01-01

    Experiments are reported on the uni-site catalysis and the transition from uni-site to multi-site catalysis with bovine heart mitochondrial F1-ATPase. The very slow uni-site ATP hydrolysis is shown to occur without tightly bound nucleotides present and with or without Pi in the buffer. Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates. They provide rate constant information, and show that an apparent Km for a second site of about 2 microM and Vmax of 10 s-1, as suggested by others, is not operative. Careful initial velocity measurements also eliminate other suggested Km values and are consistent with bi-site activation to near maximal hydrolysis rates, with a Km of about 130 microM and Vmax of about 700 s-1. However, the results do not eliminate the possibility of additional 'hidden' Km values with similar Vmax:Km ratios. Recent data on competition between TNP-ATP and ATP revealed a third catalytic site for ATP in the millimolar concentration range. This result, and those reported in the present paper, allow the conclusion that the mitochondrial F1-ATPase can attain near maximal activity in bi-site catalysis. Our data also add to the evidence that a recent claim, that the mitochondrial F1-ATPase does not show catalytic site cooperativity, is invalid. PMID:9480927

  18. Features of the Built Environment Related to Physical Activity Friendliness and Children’s Obesity and Other Risk Factors

    PubMed Central

    Taylor, Wendell C.; Upchurch, Sandra L.; Brosnan, Christine A.; Selwyn, Beatrice J.; Nguyen, Thong Q.; Villagomez, Evangelina Trejo; Meininger, Janet C.

    2014-01-01

    Objectives We investigated the relationships among environmental features of physical activity friendliness, socioeconomic indicators, and prevalence of obesity (BMI status), central adiposity (waist circumference, waist-height ratio), and hypertension. Design The design was cross-sectional; the study was correlational. Sample The sample was 911 kindergarteners through sixth graders from three schools in an urban school district residing in 13 designated neighborhoods. Measurements Data from walking environmental community audits, census data for socioeconomic indicators, body mass index, waist circumference, waist-height ratio, and blood pressure were analyzed. A modified Alfonzo’s Hierarchy of Walking Needs model was the conceptual framework for environmental features (i.e., accessibility, safety, comfort, and pleasurability) related to physical activity. Results Accessibility was significantly and negatively correlated with prevalence of obesity and with prevalence of a waist-height ratio > 0.50. When neighborhood education was controlled, and when both neighborhood education and poverty were controlled with partial correlational analysis, comfort features of a walking environment were significantly and positively related to prevalence of obesity. When poverty was controlled with partial correlation, accessibility was significantly and negatively correlated with prevalence of waist-height ratio > 0.50. Conclusions The built environment merits further research to promote physical activity and stem the obesity epidemic in children. Our approach can be a useful framework for future research. PMID:25112374

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

    SciTech Connect

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

    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.

  20. Modified Active Site Coordination in a Clinical Mutant of Sulfite Oxidase

    SciTech Connect

    Doonan, C.J.; Wilson, H.L.; Rajagopalan, K.V.; Garrett, R.M.; Bennett, B.; Prince, R.C.; George, G.N.

    2009-06-02

    The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme sulfite oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.

  1. Basaltic lava flows covering active aeolian dunes in the Paraná Basin in southern Brazil: Features and emplacement aspects

    NASA Astrophysics Data System (ADS)

    Waichel, Breno L.; Scherer, Claiton M. S.; Frank, Heinrich T.

    2008-03-01

    Burial of active aeolian dunes by lava flows can preserve the morphology of the dunes and generate diverse features related to interaction between unconsolidated sediments and lavas. In the study area, located in southern Brazil, burial of aeolian deposits by Cretaceous basaltic lava flows completely preserved dunes, and generate sand-deformation features, sand diapirs and peperite-like breccia. The preserved dunes are crescentic and linear at the main contact with basalts, and smaller crescentic where interlayered with lavas. The various feature types formed on sediment surfaces by the advance of the flows reflect the emplacement style of the lavas which are compound pahoehoe type. Four feature types can be recognized: (a) type 1 features are related to the advance of sheet flows in dune-interdune areas with slopes > 5°, (b) type 2 is formed where the lava flows advance in lobes and climb the stoss slope of crescentic dunes (slopes 8-12°), (c) type 3 is generated by toes that descend the face of linear dunes (slopes 17-23°) and (d) type 4 occurs when lava lobes descend the stoss slope of crescentic dunes (slopes 10-15°). The direction of the flows, the disposition and morphology of the dunes and the ground slope are the main factors controlling formation of the features. The injection of unconsolidated sand in lava lobes forms diapirs and peperite-like breccias. Sand diapirs occur at the basal portion of lobes where the lava was more solidified. Peperite-like breccias occur in the inner portion where lava was more plastic, favoring the mingling of the components. The generation of both features is related to a mechanical process: the weight of the lava causes the injection of sand into the lava and the warming of the air in the pores of the sand facilitates this process. The lava-sediment interaction features presented here are consistent with previous reports of basalt lavas with unconsolidated arid sediments, and additional new sand-deformation features

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

    PubMed

    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

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

    PubMed Central

    Petrosino, J F; Palzkill, T

    1996-01-01

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

  4. Physical activity advertisements that feature daily well-being improve autonomy and body image in overweight women but not men.

    PubMed

    Segar, Michelle L; Updegraff, John A; Zikmund-Fisher, Brian J; Richardson, Caroline R

    2012-01-01

    The reasons for exercising that are featured in health communications brand exercise and socialize individuals about why they should be physically active. Discovering which reasons for exercising are associated with high-quality motivation and behavioral regulation is essential to promoting physical activity and weight control that can be sustained over time. This study investigates whether framing physical activity in advertisements featuring distinct types of goals differentially influences body image and behavioral regulations based on self-determination theory among overweight and obese individuals. Using a three-arm randomized trial, overweight and obese women and men (aged 40-60 yr, n = 1690) read one of three ads framing physical activity as a way to achieve (1) better health, (2) weight loss, or (3) daily well-being. Framing effects were estimated in an ANOVA model with pairwise comparisons using the Bonferroni correction. This study showed that there are immediate framing effects on physical activity behavioral regulations and body image from reading a one-page advertisement about physical activity and that gender and BMI moderate these effects. Framing physical activity as a way to enhance daily well-being positively influenced participants' perceptions about the experience of being physically active and enhanced body image among overweight women, but not men. The experiment had less impact among the obese study participants compared to those who were overweight. These findings support a growing body of research suggesting that, compared to weight loss, framing physical activity for daily well-being is a better gain-frame message for overweight women in midlife. PMID:22701782

  5. Phenomenological features of mortality and morbidity dynamics in Tomsk versus heliogeophysical activity

    NASA Astrophysics Data System (ADS)

    Borodin, A. S.; Tuzhilkin, D. A.; Gudina, M. V.; Vladimirsky, B. M.

    2015-12-01

    The influence of heliogeophysical activity on the morbidity and mortality of the population in Tomsk is studied epidemiologically on the basis of regional data. The biological effectiveness of heliogeophysical factors selected on the basis of the Karhunen-Loeve method from epidemiological data on the morbidity and mortality in Tomsk in 1990-2008 is estimated. An analysis of the impact of variations in heliogeophysical activity on morbidity and mortality (according to the International Statistical Classification of Diseases and Related Health, ICD-10) showed the existence of common factors within different nosological classes that reliably correlate with the major components of variations in characteristic indices of heliogeophysical activity.

  6. Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents

    PubMed Central

    Hudson, Elton P; Eppler, Ross K; Beaudoin, Julianne M; Dordick, Jonathan S; Reimer, Jeffrey A; Clark, Douglas S

    2009-01-01

    The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, aw. The apparent turnover number kcatapp in these systems ranged from 0.2 to 80 s−1 and increased 11-fold in isooctane and up to 50-fold in tetrahydrofuran with increasing aw. 19F-NMR relaxation experiments employing an active-site inhibitor were used to assess the dependence of active-site motions on aw. The rates of NMR-derived fast (k > 107 s−1) and slow (k < 104 s−1) active-site motions increased in both solvents upon hydration, but only the slow motions correlated with kcat. The 19F chemical shift was a sensitive probe of the local electronic environment and provided an empirical measure of the active-site dielectric constant εas, which increased with hydration to εas ≈ 13 in each solvent. In both solvents the transition state free energy data and εas followed Kirkwood’s model for the continuum solvation of a dipole, indicating that water also enhanced catalysis by altering the active-site’s electronic environment and increasing its polarity to better stabilize the transition state. These results reveal that favorable dynamic and electrostatic effects both contribute to accelerated catalysis by solubilized subtilisin Carlsberg upon hydration in organic solvents. PMID:19317505

  7. Acylpeptide hydrolase: inhibitors and some active site residues of the human enzyme.

    PubMed

    Scaloni, A; Jones, W M; Barra, D; Pospischil, M; Sassa, S; Popowicz, A; Manning, L R; Schneewind, O; Manning, J M

    1992-02-25

    Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions. PMID:1740429

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

    PubMed

    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

  9. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

    SciTech Connect

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-27

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

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

    PubMed Central

    Wang, Lu; Fried, Stephen D.; Boxer, Steven G.; Markland, Thomas E.

    2014-01-01

    Enzymes use 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. PMID:25503367

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

    PubMed

    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

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

    PubMed Central

    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 Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) 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, Thr890, Ser893, Thr894 and Thr899, 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

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

    PubMed

    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

    NASA Astrophysics Data System (ADS)

    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

    SciTech Connect

    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. Activity-dependent labeling of oxygenase enzymes in a trichloroethene-contaminated groundwater site.

    PubMed

    Lee, M Hope; Clingenpeel, Scott C; Leiser, Owen P; Wymore, Ryan A; Sorenson, Kent S; Watwood, Mary E

    2008-05-01

    A variety of naturally occurring bacteria produce enzymes that cometabolically degrade trichloroethene (TCE), including organisms with aerobic oxygenases. Groundwater contaminated with TCE was collected from the aerobic region of the Test Area North site of the Idaho National Laboratory. Samples were evaluated with enzyme activity probes, and resulted in measurable detection of toluene oxygenase activity (6-79% of the total microbial cells). Wells from both inside and outside contaminated plume showed activity. Toluene oxygenase-specific PCR primers determined that toluene-degrading genes were present in all groundwater samples evaluated. In addition, bacterial isolates were obtained and possessed toluene oxygenase enzymes, demonstrated activity, and were dominated by the phylotype Pseudomonas. This study demonstrated, through the use of enzymatic probes and oxygenase gene identification, that indigenous microorganisms at a contaminated site were cometabolically active. Documentation such as this can be used to substantiate observations of natural attenuation of TCE-contaminated groundwater plumes. PMID:17904715

  17. An Analysis of Activities in Saudi Arabian Middle School Science Textbooks and Workbooks for the Inclusion of Essential Features of Inquiry

    NASA Astrophysics Data System (ADS)

    Aldahmash, Abdulwali H.; Mansour, Nasser S.; Alshamrani, Saeed M.; Almohi, Saeed

    2016-01-01

    This study examines Saudi Arabian middle school science textbooks' coverage of the essential features of scientific inquiry. All activities in the middle school science textbooks and workbooks were analyzed by using the scientific inquiry `essential features' rubric. The results indicated that the essential features are included in about 59 % of the analyzed science activities. However, feature 2, `making learner give priority to evidence in responding to questions' and feature 3, `allowing learner to formulate explanations from evidence' appeared more frequently than the other three features (feature 1: engaging learner in scientifically oriented questions, feature 4: helping learner connect explanations to scientific knowledge, and feature 5: helping learner communicate and justify explanations to others), whether in the activities as a whole, or in the activities included in each of the four science domains (physical science, Earth science, life science and chemistry). These features are represented in almost all activities. This means that almost all activities in the middle school science textbooks and the workbooks include features 2 and 3. Meanwhile, the mean level of inclusion of the five essential features of scientific inquiry found in the middle school science textbooks and workbooks as a whole is 2.55. However, results found for features 1, 4, 5 and for in-level inclusion of the inquiry features in each of the science domains indicate that the inclusion of the essential inquiry features is teacher-centred. As a result, neither science textbooks nor workbooks provide students with the opportunity or encouragement to develop their inquiry skills. Consequently, the results suggest important directions for educational administrators and policy-makers in the preparation and use of science educational content.

  18. DNA damage processing by human 8-oxoguanine-DNA glycosylase mutants with the occluded active site.

    PubMed

    Lukina, Maria V; Popov, Alexander V; Koval, Vladimir V; Vorobjev, Yuri N; Fedorova, Olga S; Zharkov, Dmitry O

    2013-10-01

    8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation. PMID:23955443

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

    SciTech Connect

    Fitzner, R.E.; Weiss, S.G.; Stegen, J.A.

    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.

  20. A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

    SciTech Connect

    Somarowthu, Srinivas; Brodkin, Heather R.; D’Aquino, J. Alejandro; Ringe, Dagmar; Ondrechen, Mary Jo; Beuning, Penny J.

    2012-07-11

    Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

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

    PubMed

    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

  2. Digital database of mining-related features at selected historic and active phosphate mines, Bannock, Bear Lake, Bingham, and Caribou counties, Idaho

    USGS Publications Warehouse

    Causey, J. Douglas; Moyle, Phillip R.

    2001-01-01

    This report provides a description of data and processes used to produce a spatial database that delineates mining-related features in areas of historic and active phosphate mining in the core of the southeastern Idaho phosphate resource area. The data have varying degrees of accuracy and attribution detail. Classification of areas by type of mining-related activity at active mines is generally detailed; however, the spatial coverage does not differentiate mining-related surface disturbance features at many of the closed or inactive mines. Nineteen phosphate mine sites are included in the study. A total of 5,728 hc (14,154 ac), or more than 57 km2 (22 mi2), of phosphate mining-related surface disturbance are documented in the spatial coverage of the core of the southeast Idaho phosphate resource area. The study includes 4 active phosphate mines—Dry Valley, Enoch Valley, Rasmussen Ridge, and Smoky Canyon—and 15 historic phosphate mines—Ballard, Champ, Conda, Diamond Gulch, Gay, Georgetown Canyon, Henry, Home Canyon, Lanes Creek, Maybe Canyon, Mountain Fuel, Trail Canyon, Rattlesnake Canyon, Waterloo, and Wooley Valley. Spatial data on the inactive historic mines is relatively up-to-date; however, spatially described areas for active mines are based on digital maps prepared in early 1999. The inactive Gay mine has the largest total area of disturbance: 1,917 hc (4,736 ac) or about 19 km2 (7.4 mi2). It encompasses over three times the disturbance area of the next largest mine, the Conda mine with 607 hc (1,504 ac), and it is nearly four times the area of the Smoky Canyon mine, the largest of the active mines with 497 hc (1,228 ac). The wide range of phosphate mining-related surface disturbance features (approximately 80) were reduced to 13 types or features used in this study—adit and pit, backfilled mine pit, facilities, mine pit, ore stockpile, railroad, road, sediment catchment, tailings or tailings pond, topsoil stockpile, water reservoir, and disturbed

  3. Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus kandleri.

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

    Aminoacyl tRNA synthetases (aaRSs) carry out the first step of protein biosynthesis. Several aaRSs are multimeric, and coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and accurate aminoacylation. However, important lacunae of understanding exist concerning the conformational dynamics of multimeric aaRSs. Questions remained unanswered pertaining to the dynamics of the active site. Little is known concerning the conformational dynamics of the active sites in response to the substrate binding, reorganization of the catalytic residues around reactants, time-dependent changes at the reaction center, which are essential for facilitating the nucleophilic attack, and interactions at the interface of neighboring monomers. In the present work, we carried out all-atom molecular dynamics simulation of dimeric (mk)SerRS from Methanopyrus kandleri bound with tRNA using an explicit solvent system. Two dimeric states of seryl tRNA synthetase (open, substrate bound, and adenylate bound) and two monomeric states (open and substrate bound) are simulated with bound tRNA. The aim is to understand the conformational dynamics of (mk)SerRS during its reaction cycle. While the present results provide a clear dynamical perspective of the active sites of (mk)SerRS, they corroborate with the results from the time-averaged experimental data such as crystallographic and mutation analysis of methanogenic SerRS from M. kandleri and M. barkeri. It is observed from the present simulation that the motif 2 loop gates the active site and its Glu351 and Arg360 stabilizes ATP in a bent state favorable for nucleophilic attack. The flexibility of the walls of the active site gradually reduces near reaction center, which is a more organized region compared to the lid region. The motif 2 loop anchors Ser and ATP using Arg349 in a hydrogen bonded geometry crucial for nucleophilic attack and favorably influences the electrostatic potential at the

  4. Monitoring of geological activity on astronomical sites of the Canary Islands, Hawaii, and Chile

    NASA Astrophysics Data System (ADS)

    Eff-Darwich, Antonio; Garcia-Lorenzo, Begoña; Rodriguez-Losada, Jose A.; Hernández-Gutiérrez, Luis E.; de la Nuez, Julio; Romero-Ruiz, Maria C.

    2009-09-01

    Future large and extremely large ground-based telescopes will demand stable geological settings.Remote sensing could be an unvaluable tool to analyse the impact of geological activity 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; the candidate site of Cerro Ventarrones, Chile). In this sense, the extent of lava flows, eruptive clouds or ground deformation associated to seismic and/or volcanic activity could be analysed and characterised through remote sensing.

  5. Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures

    NASA Astrophysics Data System (ADS)

    Schreiber, Andreas; Huber, Matthias C.; Cölfen, Helmut; Schiller, Stefan M.

    2015-03-01

    The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based ‘adaptors/connectors’ with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties.

  6. Molecular protein adaptor with genetically encoded interaction sites guiding the hierarchical assembly of plasmonically active nanoparticle architectures.

    PubMed

    Schreiber, Andreas; Huber, Matthias C; Cölfen, Helmut; Schiller, Stefan M

    2015-01-01

    The control over the defined assembly of nano-objects with nm-precision is important to create systems and materials with enhanced properties, for example, metamaterials. In nature, the precise assembly of inorganic nano-objects with unique features, for example, magnetosomes, is accomplished by efficient and reliable recognition schemes involving protein effectors. Here we present a molecular approach using protein-based 'adaptors/connectors' with genetically encoded interaction sites to guide the assembly and functionality of different plasmonically active gold nanoparticle architectures (AuNP). The interaction of the defined geometricaly shaped protein adaptors with the AuNP induces the self-assembly of nanoarchitectures ranging from AuNP encapsulation to one-dimensional chain-like structures, complex networks and stars. Synthetic biology and bionanotechnology are applied to co-translationally encode unnatural amino acids as additional site-specific modification sites to generate functionalized biohybrid nanoarchitectures. This protein adaptor-based nano-object assembly approach might be expanded to other inorganic nano-objects creating biohybrid materials with unique electronic, photonic, plasmonic and magnetic properties. PMID:25813537

  7. Ordinary and Activated Bone Grafts: Applied Classification and the Main Features

    PubMed Central

    Deev, R. V.; Drobyshev, A. Y.; Bozo, I. Y.; Isaev, A. A.

    2015-01-01

    Bone grafts are medical devices that are in high demand in clinical practice for substitution of bone defects and recovery of atrophic bone regions. Based on the analysis of the modern groups of bone grafts, the particularities of their composition, the mechanisms of their biological effects, and their therapeutic indications, applicable classification was proposed that separates the bone substitutes into “ordinary” and “activated.” The main differential criterion is the presence of biologically active components in the material that are standardized by qualitative and quantitative parameters: growth factors, cells, or gene constructions encoding growth factors. The pronounced osteoinductive and (or) osteogenic properties of activated osteoplastic materials allow drawing upon their efficacy in the substitution of large bone defects. PMID:26649300

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

    PubMed Central

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

    2015-01-01

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

  9. Active-Site Monovalent Cations Revealed in a 1.55 Å Resolution Hammerhead Ribozyme Structure

    PubMed Central

    Anderson, Michael; Schultz, Eric P.; Martick, Monika; Scott, William G.

    2013-01-01

    We have obtained a 1.55 Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni in conditions that permit detailed observations of Na+ ion binding in the ribozyme's active site. At least two such Na+ ions are observed. The first Na+ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical to that previously observed for divalent cations. A second Na+ ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na+, but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na+ directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest resolution ribozyme structure in the protein data bank. PMID:23711504

  10. Tuned by metals: the TET peptidase activity is controlled by 3 metal binding sites

    PubMed Central

    Colombo, Matteo; Girard, Eric; Franzetti, Bruno

    2016-01-01

    TET aminopeptidases are dodecameric particles shared in the three life domains involved in various biological processes, from carbon source provider in archaea to eye-pressure regulation in humans. Each subunit contains a dinuclear metal site (M1 and M2) responsible for the enzyme catalytic activity. However, the role of each metal ion is still uncharacterized. Noteworthy, while mesophilic TETs are activated by Mn2+, hyperthermophilic TETs prefers Co2+. Here, by means of anomalous x-ray crystallography and enzyme kinetics measurements of the TET3 aminopeptidase from the hyperthermophilic organism Pyrococcus furiosus (PfTET3), we show that M2 hosts the catalytic activity of the enzyme, while M1 stabilizes the TET3 quaternary structure and controls the active site flexibility in a temperature dependent manner. A new third metal site (M3) was found in the substrate binding pocket, modulating the PfTET3 substrate preferences. These data show that TET activity is tuned by the molecular interplay among three metal sites. PMID:26853450

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Small activating RNA binds to the genomic target site in a seed-region-dependent manner

    PubMed Central

    Meng, Xing; Jiang, Qian; Chang, Nannan; Wang, Xiaoxia; Liu, Chujun; Xiong, Jingwei; Cao, Huiqing; Liang, Zicai

    2016-01-01

    RNA activation (RNAa) is the upregulation of gene expression by small activating RNAs (saRNAs). In order to investigate the mechanism by which saRNAs act in RNAa, we used the progesterone receptor (PR) gene as a model, established a panel of effective saRNAs and assessed the involvement of the sense and antisense strands of saRNA in RNAa. All active saRNAs had their antisense strand effectively incorporated into Ago2, whereas such consistency did not occur for the sense strand. Using a distal hotspot for saRNA targeting at 1.6-kb upstream from the PR transcription start site, we further established that gene activation mediated by saRNA depended on the complementarity of the 5′ region of the antisense strand, and that such activity was largely abolished by mutations in this region of the saRNA. We found markedly reduced RNAa effects when we created mutations in the genomic target site of saRNA PR-1611, thus providing evidence that RNAa depends on the integrity of the DNA target. We further demonstrated that this saRNA bound the target site on promoter DNA. These results demonstrated that saRNAs work via an on-site mechanism by binding to target genomic DNA in a seed-region-dependent manner, reminiscent of miRNA-like target recognition. PMID:26873922

  13. A Ty1 Reverse Transcriptase Active-Site Aspartate Mutation Blocks Transposition but Not Polymerization†

    PubMed Central

    Uzun, Ozcan; Gabriel, Abram

    2001-01-01

    Reverse transcriptases (RTs) are found in a wide variety of mobile genetic elements including viruses, retrotransposons, and infectious organellar introns. An invariant triad of aspartates is thought to be required for the catalytic function of RTs. We generated RT mutants in the yeast retrotransposon Ty1, changing each of these active-site aspartates to asparagine or glutamate. All but one of the mutants lacked detectable polymerase activity. The novel exception, D211N, retained near wild-type in vitro polymerase activity within virus-like particles but failed to carry out in vivo transposition. For this mutant, minus-strand synthesis is impaired and formation of the plus-strand strong-stop intermediate is eliminated. Intragenic second-site suppressor mutations of the transposition defect map to the RNase H domain of the enzyme. Our results demonstrate that one of the three active-site aspartates in a retrotransposon RT is not catalytically critical. This implies a basic difference in the polymerase active-site geometry of Ty1 and human immunodeficiency virus RT and shows that subtle mutations in one domain can cause dramatic functional effects on a distant domain of the same enzyme. PMID:11413300

  14. FIBRONECTIN-FRAGMENTATION IS A FEATURE OF BOTH PERIODONTAL DISEASE SITES AND DIABETIC FOOT AND LEG WOUNDS AND MODIFIES CELL BEHAVIOR

    PubMed Central

    Stanley, Corey M.; Wang, Yao; Pal, Sanjay; Klebe, Robert J.; Harkless, Lawrence B.; Xu, Xiaoping; Chen, Zhihua; Steffensen, Bjorn

    2009-01-01

    Background: Fibronectin (FN) undergoes fragmentation in periodontal disease sites and in poorly-healing diabetic wounds. The biological effects of FN fragments on wound healing remain unresolved. This study characterized the pattern of FN fragmentation and its effects on cellular behavior compared to intact FN. Methods: Polyclonal antibodies were raised against FN and three defined recombinant segments of FN and used to analyze gingival crevicular fluid (GCF) from periodontal disease sites in systemically healthy and diabetic patients as well as chronic leg and foot wound exudates from patients with diabetes. Subsequently, the behavior of human gingival fibroblasts (hGF) and HT1080 reference cells were analyzed by measuring cell attachment, migration, and chemotaxis in the presence of intact FN or recombinant FN fragments. Results: FN fragmentation was evident in fluids from periodontal disease sites and diabetic leg and foot wounds. However, no fragmentation pattern distinguished systemically healthy patients from patients with diabetes. Both hGF and HT1080 cells required significantly higher concentrations of FN fragments to achieve attachment comparable to intact FN. Cells cultured on FN fragments also were morphologically very different from cells cultured on full-length FN. Migration was reduced for hGF cultured on FN fragments relative to full-length FN. In contrast, FN fragments increased HT1080 fibrosarcoma cell migration over intact FN. Conclusions: These experiments demonstrated that FN fragmentation is a prominent feature of both periodontal and chronic leg and foot wounds in diabetes. Furthermore, cell culture assays confirmed the hypothesis that exposure to defined FN fragments significantly alters cell behavior. PMID:18454665

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Role of methionine in the active site of alpha-galactosidase from Trichoderma reesei.

    PubMed Central

    Kachurin, A M; Golubev, A M; Geisow, M M; Veselkina, O S; Isaeva-Ivanova, L S; Neustroev, K N

    1995-01-01

    alpha-Galactosidase from Trichoderma reesei when treated with H2O2 shows a 12-fold increase in activity towards p-nitrophenyl alpha-D-galactopyranoside. A similar effect is produced by the treatment of alpha-galactosidase with other non-specific oxidants: NaIO4, KMnO4 and K4S4O8. In addition to the increase in activity, the Michaelis constant rises from 0.2 to 1.4 mM, the temperature coefficient decreases by a factor of 1.5 and the pH-activity curve falls off sharply with increasing pH. Galactose (a competitive inhibitor of alpha-galactosidase; Ki 0.09 mM for the native enzyme at pH 4.4) effectively inhibits oxidative activation of the enzyme, because the observed activity changes are related to oxidation of the catalytically important methionine in the active site. NMR measurements and amino acid analysis show that oxidation to methionine sulphoxide of one of five methionines is sufficient to activate alpha-galactosidase. Binding of galactose prevents this. Oxidative activation does not lead to conversion of other H2O2-sensitive amino acid residues, such as histidine, tyrosine, tryptophan and cysteine. The catalytically important cysteine thiol group is quantitatively titrated after protein oxidative activation. Further oxidation of methionines (up to four of five residues) can be achieved by increasing the oxidation time and/or by prior denaturation of the protein. Obviously, a methionine located in the active site of alpha-galactosidase is more accessible. The oxidative-activation phenomenon can be explained by a conformational change in the active site as a result of conversion of non-polar methionine into polar methionine sulphoxide. Images Figure 10 PMID:8948456

  17. Incidental and context-responsive activation of structure- and function-based action features during object identification.

    PubMed

    Lee, Chia-lin; Middleton, Erica; Mirman, Daniel; Kalénine, Solène; Buxbaum, Laurel J

    2013-02-01

    Previous studies suggest that action representations are activated during object processing, even when task-irrelevant. In addition, there is evidence that lexical-semantic context may affect such activation during object processing. Finally, prior work from our laboratory and others indicates that function-based ("use") and structure-based ("move") action subtypes may differ in their activation characteristics. Most studies assessing such effects, however, have required manual object-relevant motor responses, thereby plausibly influencing the activation of action representations. The present work uses eyetracking and a Visual World Paradigm task without object-relevant actions to assess the time course of activation of action representations, as well as their responsiveness to lexical-semantic context. In two experiments, participants heard a target word and selected its referent from an array of four objects. Gaze fixations on nontarget objects signal activation of features shared between targets and nontargets. The experiments assessed activation of structure-based (Experiment 1) or function-based (Experiment 2) distractors, using neutral sentences ("S/he saw the....") or sentences with a relevant action verb (Experiment 1: "S/he picked up the...."; Experiment 2: "S/he used the...."). We observed task-irrelevant activations of action information in both experiments. In neutral contexts, structure-based activation was relatively faster-rising but more transient than function-based activation. Additionally, action verb contexts reliably modified patterns of activation in both Experiments. These data provide fine-grained information about the dynamics of activation of function-based and structure-based actions in neutral and action-relevant contexts, in support of the "Two Action System" model of object and action processing (e.g., Buxbaum & Kalénine, 2010). PMID:22390294

  18. Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site.

    PubMed

    Gajadeera, Chathurada S; Zhang, Xinyi; Wei, Yinan; Tsodikov, Oleg V

    2015-02-01

    Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg(2+)-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn(2+)-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn(2+) at 2.1Å resolution. The active site contains two catalytic Mn(2+) binding sites, each half-occupied, reconciling the previously observed 1:1 Mn(2+):enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295-298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme. PMID:25576794

  19. NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site

    PubMed Central

    Hoffmann, Bernd; Mitchell, G. Thomas; Gendron, Patrick; Major, François; Andersen, Angela A.; Collins, Richard A.; Legault, Pascale

    2003-01-01

    Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg2+ binding sites in the active internal loop. One of the Mg2+ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program mc-search, we identified the active internal loop fold in other RNA structures. In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme. PMID:12782785

  20. Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

    PubMed Central

    Sayer, Christopher; Finnigan, William; Isupov, Michail N.; Levisson, Mark; Kengen, Servé W. M.; van der Oost, John; Harmer, Nicholas J.; Littlechild, Jennifer A.

    2016-01-01

    A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions. PMID:27160974

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

    SciTech Connect

    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.

  2. ActiveTutor: Towards More Adaptive Features in an E-Learning Framework

    ERIC Educational Resources Information Center

    Fournier, Jean-Pierre; Sansonnet, Jean-Paul

    2008-01-01

    Purpose: This paper aims to sketch the emerging notion of auto-adaptive software when applied to e-learning software. Design/methodology/approach: The study and the implementation of the auto-adaptive architecture are based on the operational framework "ActiveTutor" that is used for teaching the topic of computer science programming in first-grade…

  3. Making rainfall features fun: scientific activities for teaching children aged 5-12 years

    NASA Astrophysics Data System (ADS)

    Gires, Auguste; Muller, Catherine L.; le Gueut, Marie-Agathe; Schertzer, Daniel

    2016-05-01

    Research projects now rely on an array of different channels to increase impact, including high-level scientific output, tools, and equipment, but also communication, outreach, and educational activities. This paper focuses on education for children aged 5-12 years and presents activities that aim to help them (and their teachers) grasp some of the complex underlying issues in environmental science. More generally, it helps children to become familiarized with science and scientists, with the aim to enhance scientific culture and promote careers in this field. The activities developed are focused on rainfall: (a) designing and using a disdrometer to observe the variety of drop sizes; (b) careful recording of successive dry and rainy days and reproducing patterns using a simple model based on fractal random multiplicative cascades; and (c) collaboratively writing a children's book about rainfall. These activities are discussed in the context of current state-of-the-art pedagogical practices and goals set by project funders, especially in a European Union framework.

  4. Apple Treats. BASIC Classroom Computing Featuring Hands-On Activities and Pencil/Paper Fun.

    ERIC Educational Resources Information Center

    Embry, Lynn

    Developed as an introduction to computer programming using an Apple microcomputer, this document presents teachers and students with: (1) essential vocabulary used in the BASIC language; (2) syntax; and (3) programming techniques. Simple hands-on activities designed to guide the learner through beginning BASIC programming skills and fundamental…

  5. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions

    PubMed Central

    Herter, Susanne; Kranz, David C; Turner, Nicholas J

    2015-01-01

    Summary Cytochrome P450 monooxygenases are useful biocatalysts for C–H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations. PMID:26664590

  6. Distinct features of circulating microparticles and their relationship with disease activity in inflammatory bowel disease

    PubMed Central

    Voudoukis, Evangelos; Vetsika, Eleni-Kyriaki; Giannakopoulou, Konstantina; Karmiris, Konstantinos; Theodoropoulou, Angeliki; Sfiridaki, Aekaterini; Georgoulias, Vassilis; Paspatis, Gregorios A.; Koutroubakis, Ioannis E.

    2016-01-01

    Background There is evidence that circulating microparticles (MPs) and annexin (+) platelet-derived MPs (PDMPs) are increased in inflammatory bowel disease (IBD). The aim of our study was to characterize the abundance, origin, and annexin V binding of MPs in patients with IBD and correlate them with the disease characteristics. Methods Case-control study of 46 IBD patients (23 Crohn’s disease, 23 ulcerative colitis) and 40 matched healthy controls (HC). MPs were divided according to annexin V binding, their origin was estimated based on specific cell membrane markers in plasma samples and their number was calculated via flow cytometry. Clinical and laboratory activity indices were also analyzed. Results Annexin (-) PDMPs (P=0.0004), total (P=0.04) and annexin (+) monocyte-derived MPs (P=0.02) were increased and annexin (-) total MPs (P=0.0007) were decreased in IBD patients compared to HC. The annexin (+)/(-) ratio of all MP types were significantly elevated in IBD patients compared to HC (P<0.003). IBD patients with active disease displayed elevated total and annexin (+) total MPs, total, annexin (+) and (-) PDMPs compared with those in remission (P<0.05). Annexin (-) PDMPs were considerably increased in IBD patients with active compared to those with inactive disease (P=0.0013). Total and annexin (-) PDMPs were significantly correlated with most of the disease activity indices (P<0.05). Conclusion The majority of circulating MPs, their counterparts and particularly annexin (-) PDMPs are increased in active IBD patients. Annexin (+)/(-) ratio proved to be the most reliable distinctive MP index between HC and IBD patients. PMID:27065731

  7. Metabolic Features of Protochlamydia amoebophila Elementary Bodies – A Link between Activity and Infectivity in Chlamydiae

    PubMed Central

    Watzka, Margarete; Wultsch, Anna; Tziotis, Dimitrios; Montanaro, Jacqueline; Richter, Andreas; Schmitt-Kopplin, Philippe; Horn, Matthias

    2013-01-01

    The Chlamydiae are a highly successful group of obligate intracellular bacteria, whose members are remarkably diverse, ranging from major pathogens of humans and animals to symbionts of ubiquitous protozoa. While their infective developmental stage, the elementary body (EB), has long been accepted to be completely metabolically inert, it has recently been shown to sustain some activities, including uptake of amino acids and protein biosynthesis. In the current study, we performed an in-depth characterization of the metabolic capabilities of EBs of the amoeba symbiont Protochlamydia amoebophila. A combined metabolomics approach, including fluorescence microscopy-based assays, isotope-ratio mass spectrometry (IRMS), ion cyclotron resonance Fourier transform mass spectrometry (ICR/FT-MS), and ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was conducted, with a particular focus on the central carbon metabolism. In addition, the effect of nutrient deprivation on chlamydial infectivity was analyzed. Our investigations revealed that host-free P. amoebophila EBs maintain respiratory activity and metabolize D-glucose, including substrate uptake as well as host-free synthesis of labeled metabolites and release of labeled CO2 from 13C-labeled D-glucose. The pentose phosphate pathway was identified as major route of D-glucose catabolism and host-independent activity of the tricarboxylic acid (TCA) cycle was observed. Our data strongly suggest anabolic reactions in P. amoebophila EBs and demonstrate that under the applied conditions D-glucose availability is essential to sustain metabolic activity. Replacement of this substrate by L-glucose, a non-metabolizable sugar, led to a rapid decline in the number of infectious particles. Likewise, infectivity of Chlamydia trachomatis, a major human pathogen, also declined more rapidly in the absence of nutrients. Collectively, these findings demonstrate that D-glucose is utilized by P. amoebophila EBs and provide

  8. Metabolic features of Protochlamydia amoebophila elementary bodies--a link between activity and infectivity in Chlamydiae.

    PubMed

    Sixt, Barbara S; Siegl, Alexander; Müller, Constanze; Watzka, Margarete; Wultsch, Anna; Tziotis, Dimitrios; Montanaro, Jacqueline; Richter, Andreas; Schmitt-Kopplin, Philippe; Horn, Matthias

    2013-01-01

    The Chlamydiae are a highly successful group of obligate intracellular bacteria, whose members are remarkably diverse, ranging from major pathogens of humans and animals to symbionts of ubiquitous protozoa. While their infective developmental stage, the elementary body (EB), has long been accepted to be completely metabolically inert, it has recently been shown to sustain some activities, including uptake of amino acids and protein biosynthesis. In the current study, we performed an in-depth characterization of the metabolic capabilities of EBs of the amoeba symbiont Protochlamydia amoebophila. A combined metabolomics approach, including fluorescence microscopy-based assays, isotope-ratio mass spectrometry (IRMS), ion cyclotron resonance Fourier transform mass spectrometry (ICR/FT-MS), and ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was conducted, with a particular focus on the central carbon metabolism. In addition, the effect of nutrient deprivation on chlamydial infectivity was analyzed. Our investigations revealed that host-free P. amoebophila EBs maintain respiratory activity and metabolize D-glucose, including substrate uptake as well as host-free synthesis of labeled metabolites and release of labeled CO2 from (13)C-labeled D-glucose. The pentose phosphate pathway was identified as major route of D-glucose catabolism and host-independent activity of the tricarboxylic acid (TCA) cycle was observed. Our data strongly suggest anabolic reactions in P. amoebophila EBs and demonstrate that under the applied conditions D-glucose availability is essential to sustain metabolic activity. Replacement of this substrate by L-glucose, a non-metabolizable sugar, led to a rapid decline in the number of infectious particles. Likewise, infectivity of Chlamydia trachomatis, a major human pathogen, also declined more rapidly in the absence of nutrients. Collectively, these findings demonstrate that D-glucose is utilized by P. amoebophila EBs and provide

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

    PubMed

    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

  10. CO Oxidation on Au/TiO2: Condition-Dependent Active Sites and Mechanistic Pathways.

    PubMed

    Wang, Yang-Gang; Cantu, David C; Lee, Mal-Soon; Li, Jun; Glezakou, Vassiliki-Alexandra; Rousseau, Roger

    2016-08-24

    We present results of ab initio electronic structure and molecular dynamics simulations (AIMD), as well as a microkinetic model of CO oxidation catalyzed by TiO2 supported Au nanocatalysts. A coverage-dependent microkinetic analysis, based on energetics obtained with density functional methods, shows that the dominant kinetic pathway, activated oxygen species, and catalytic active sites are all strongly depended on both temperature and oxygen partial pressure. Under oxidizing conditions and T < 400 K, the prevalent pathway involves a dynamic single atom catalytic mechanism. This reaction is catalyzed by a transient Au-CO species that migrates from the Au-cluster onto a surface oxygen adatom. It subsequently reacts with the TiO2 support via a Mars van Krevelen mechanism to form CO2 and finally the Au atom reintegrates back into the gold cluster to complete the catalytic cycle. At 300 ≤ T ≤ 600 K, oxygen-bound single Oad-Au(+)-CO sites and the perimeter Au-sites of the nanoparticle work in tandem to optimally catalyze the reaction. Above 600 K, a variety of alternate pathways associated with both single-atom and the perimeter sites of the Au nanoparticle are found to be active. Under low oxygen pressures, Oad-Au(+)-CO species can be a source of catalyst deactivation and the dominant pathway involves only Au-perimeter sites. A detailed comparison of the current model and the existing literature resolves many apparent inconsistencies in the mechanistic interpretations. PMID:27480512

  11. Investigation of the active site and the conformational stability of nucleoside diphosphate kinase by site-directed mutagenesis.

    PubMed

    Tepper, A D; Dammann, H; Bominaar, A A; Véron, M

    1994-12-23

    Nucleoside-diphosphate kinase (EC 2.7.4.6) catalyzes phosphate exchange between nucleoside triphosphates and nucleoside diphosphates. Its 17 kDa subunits are highly conserved throughout evolution in both sequence and tertiary structure. Using site-directed mutagenesis we investigated the function of 8 amino acids (Lys16, Tyr56, Arg92, Thr98, Arg109, Asn119, Ser124, and Glu133) that are totally conserved among all nucleoside diphosphate kinases known to date. The mutant proteins all show decreased specific activity and support roles for these residues in catalysis, substrate binding, or both, as was previously proposed on the basis of the x-ray structure (Moréra, S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Véron, M., and Janin, J. (1994) Biochemistry 33, 459-467). Furthermore, residues Lys16, Arg109, and Asn 119 were identified to play important roles in conformational stability or subunit interactions. We show that Lys16 and Asn119 form a rigid structure that is important for enzymatic function and that Arg109, known to interact with the phosphate moiety of the substrate, also plays an important role in subunit association. The dual roles of Lys16, Arg109, and Asn119 in both substrate binding and subunit assembly provide further evidence for a functional coupling between catalytic activity and quaternary structure in nucleoside diphosphate kinase. PMID:7798215

  12. Enhancing magnetic nanoparticle-based DNA transfection: Intracellular-active cassette features

    NASA Astrophysics Data System (ADS)

    Vernon, Matthew Martin

    Efficient plasmid DNA transfection of embryonic stem cells, mesenchymal stem cells, neural cell lines and the majority of primary cell lines is a current challenge in gene therapy research. Magnetic nanoparticle-based DNA transfection is a gene vectoring technique that is promising because it is capable of outperforming most other non-viral transfection methods in terms of both transfection efficiency and cell viability. The nature of the DNA vector implemented depends on the target cell phenotype, where the particle surface chemistry and DNA binding/unbinding kinetics of the DNA carrier molecule play a critical role in the many steps required for successful gene transfection. Accordingly, Neuromag, an iron oxide/polymer nanoparticle optimized for transfection of neural phenotypes, outperforms many other nanoparticles and lipidbased DNA carriers. Up to now, improvements to nanomagnetic transfection techniques have focused mostly on particle functionalization and transfection parameter optimization (cell confluence, growth media, serum starvation, magnet oscillation parameters, etc.). None of these parameters are capable of assisting the nuclear translocation of delivered plasmid DNA once the particle-DNA complex is released from the endosome and dissociates in the cell's cytoplasm. In this study, incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid DNA confers improved nuclear translocation, demonstrating significant improvement in nanomagnetic transfection efficiency in differentiated SH-SY5Y neuroblastoma cells. Other parameters, such as days in vitro, are also found to play a role and represent potential targets for further optimization.

  13. Directing reaction pathways by catalyst active-site selection using self-assembled monolayers.

    PubMed

    Pang, Simon H; Schoenbaum, Carolyn A; Schwartz, Daniel K; Medlin, J Will

    2013-01-01

    One key route for controlling reaction selectivity in heterogeneous catalysis is to prepare catalysts that exhibit only specific types of sites required for desired product formation. Here we show that alkanethiolate self-assembled monolayers with varying surface densities can be used to tune selectivity to desired hydrogenation and hydrodeoxygenation products during the reaction of furfural on supported palladium catalysts. Vibrational spectroscopic studies demonstrate that the selectivity improvement is achieved by controlling the availability of specific sites for the hydrogenation of furfural on supported palladium catalysts through the selection of an appropriate alkanethiolate. Increasing self-assembled monolayer density by controlling the steric bulk of the organic tail ligand restricts adsorption on terrace sites and dramatically increases selectivity to desired products furfuryl alcohol and methylfuran. This technique of active-site selection simultaneously serves both to enhance selectivity and provide insight into the reaction mechanism. PMID:24025780

  14. Lessons learned from DOE site culture change activities: Implications for waste management organizations

    SciTech Connect

    Kurstedt, H.A. Jr.; Howard, E.M.; Doss, A.R.; Mallak, L.A.

    1991-01-01

    Management Systems Laboratories (MSL) has worked with the US Department of Energy (DOE) and several of its contractors as they understand and assess the DOE culture change and change the contractor culture to serve DOE's needs. Primarily, these contractors have been those whose responsibilities include starting up and operating weapons materials facilities. The number and scope of these activities have escalated and expanded to contractors at DOE sites such as Westinghouse at the Savannah River Site (SRS) in Aiken, South Carolina, EG G at the Rocky Flats Plant (RFP) in Golden, Colorado, and Westinghouse at the Feed Materials Processing Center (FMPC) in Fernald, Ohio. The point of this paper is not to compare or contrast the relative merit of one site over another. It is to show the lessons, good and bad, and use and communicate those lessons, especially those lessons transferable to other sites in similar situations. 8 refs., 1 fig.

  15. X-ray absorption spectroscopic studies of the active sites of nickel- and copper-containing metalloproteins

    SciTech Connect

    Tan, G.O.

    1993-06-01

    X-ray absorption spectroscopy (XAS) is a useful tool for obtaining structural and chemical information about the active sites of metalloproteins and metalloenzymes. Information may be obtained from both the edge region and the extended X-ray absorption fine structure (EXAFS) or post-edge region of the K-edge X-ray absorption spectrum of a metal center in a compound. The edge contains information about the valence electronic structure of the atom that absorbs the X-rays. It is possible in some systems to infer the redox state of the metal atom in question, as well as the geometry and nature of ligands connected to it, from the features in the edge in a straightforward manner. The EXAFS modulations, being produced by the backscattering of the ejected photoelectron from the atoms surrounding the metal atom, provide, when analyzed, information about the number and type of neighbouring atoms, and the distances at which they occur. In this thesis, analysis of both the edge and EXAFS regions has been used to gain information about the active sites of various metalloproteins. The metalloproteins studied were plastocyanin (Pc), laccase and nickel carbon monoxide dehydrogenase (Ni CODH). Studies of Cu(I)-imidazole compounds, related to the protein hemocyanin, are also reported here.

  16. Constrained water access to the active site of cytochrome P450 from the piezophilic bacterium Photobacterium profundum

    NASA Astrophysics Data System (ADS)

    Sineva, Elena V.; Davydov, Dmitri R.

    2010-12-01

    Living species inhabiting ocean deeps must adapt to high hydrostatic pressure. This adaptation, which must enable functioning under conditions of promoted protein hydration, is especially important for proteins such as cytochromes P450 that exhibit functionally important hydration-dehydration dynamics. Here we study the interactions of substrates with cytochrome P450-SS9, a putative fatty acid hydroxylase from the piezophilic bacterium Photobacterium profundum SS9, and characterize the protein's barotropic properties. Comparison of P450-SS9 with cytochrome P450BM-3, a mesophilic fatty acid hydroxylase, suggests that P450-SS9 is characterized by severely confined accessibility and low water occupancy of the active site. This feature may reveal a mechanism for the structural adaptation of the piezophilic enzyme. We also demonstrate that saturated and unsaturated fatty acids exert opposite effects on solvent accessibility and hydration of the active site. Modulation of the protein conformation by fatty acids is hypothesized to have an important physiological function in the piezophile.

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

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

    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

  19. Thiolactomycin inhibits D-aspartate oxidase: a novel approach to probing the active site environment.

    PubMed

    Katane, Masumi; Saitoh, Yasuaki; Hanai, Toshihiko; Sekine, Masae; Furuchi, Takemitsu; Koyama, Nobuhiro; Nakagome, Izumi; Tomoda, Hiroshi; Hirono, Shuichi; Homma, Hiroshi

    2010-10-01

    D-Aspartate oxidase (DDO) and D-amino acid oxidase (DAO) are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of D-amino acids. While several functionally and structurally important amino acid residues have been identified in the DAO protein, little is known about the structure-function relationships of DDO. In the search for a potent DDO inhibitor as a novel tool for investigating its structure-function relationships, a large number of biologically active compounds of microbial origin were screened for their ability to inhibit the enzymatic activity of mouse DDO. We discovered several compounds that inhibited the activity of mouse DDO, and one of the compounds identified, thiolactomycin (TLM), was then characterized and evaluated as a novel DDO inhibitor. TLM reversibly inhibited the activity of mouse DDO with a mixed type of inhibition more efficiently than meso-tartrate and malonate, known competitive inhibitors of mammalian DDOs. The selectivity of TLM was investigated using various DDOs and DAOs, and it was found that TLM inhibits not only DDO, but also DAO. Further experiments with apoenzymes of DDO and DAO revealed that TLM is most likely to inhibit the activities of DDO and DAO by competition with both the substrate and the coenzyme, FAD. Structural models of mouse DDO/TLM complexes supported this finding. The binding mode of TLM to DDO was validated further by site-directed mutagenesis of an active site residue, Arg-237. Collectively, our findings show that TLM is a novel, active site-directed DDO inhibitor that will be useful for elucidating the molecular details of the active site environment of DDO. PMID:20603179

  20. Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

    PubMed

    Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

    2016-07-19

    The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes

  1. Noticing relevant problem features: activating prior knowledge affects problem solving by guiding encoding

    PubMed Central

    Crooks, Noelle M.; Alibali, Martha W.

    2013-01-01

    This study investigated whether activating elements of prior knowledge can influence how problem solvers encode and solve simple mathematical equivalence problems (e.g., 3 + 4 + 5 = 3 + __). Past work has shown that such problems are difficult for elementary school students (McNeil and Alibali, 2000). One possible reason is that children's experiences in math classes may encourage them to think about equations in ways that are ultimately detrimental. Specifically, children learn a set of patterns that are potentially problematic (McNeil and Alibali, 2005a): the perceptual pattern that all equations follow an “operations = answer” format, the conceptual pattern that the equal sign means “calculate the total”, and the procedural pattern that the correct way to solve an equation is to perform all of the given operations on all of the given numbers. Upon viewing an equivalence problem, knowledge of these patterns may be reactivated, leading to incorrect problem solving. We hypothesized that these patterns may negatively affect problem solving by influencing what people encode about a problem. To test this hypothesis in children would require strengthening their misconceptions, and this could be detrimental to their mathematical development. Therefore, we tested this hypothesis in undergraduate participants. Participants completed either control tasks or tasks that activated their knowledge of the three patterns, and were then asked to reconstruct and solve a set of equivalence problems. Participants in the knowledge activation condition encoded the problems less well than control participants. They also made more errors in solving the problems, and their errors resembled the errors children make when solving equivalence problems. Moreover, encoding performance mediated the effect of knowledge activation on equivalence problem solving. Thus, one way in which experience may affect equivalence problem solving is by influencing what students encode about the

  2. Structural features and antioxidant activities of lignins from steam-exploded bamboo (Phyllostachys pubescens).

    PubMed

    Sun, Shao-Ni; Cao, Xue-Fei; Xu, Feng; Sun, Run-Cang; Jones, Gwynn Lloyd

    2014-06-25

    An environmentally friendly steam explosion process of bamboo, followed by alkali and alkaline ethanol delignification, was developed to fractionate lignins. Results showed that after steam explosion the lignins isolated showed relatively low carbohydrate contents (0.55-1.76%) and molecular weights (780-1050 g/mol). For each steam-exploded sample, alkali-extracted lignins presented higher phenolic OH values (1.41-1.82 mmol/g), p-coumaric acid to ferulic acid ratios (pCA/FA ratios 4.5-14.1), and syringyl to guaiacyl ratios (S/G ratios 5.0-8.5) than those from alkaline ethanol-extracted lignins (phenolic OH 0.85-1.35 mmol/g, pCA/FA ratios 1.6-5.2, and S/G ratios 3.5-4.8). The lignins obtained consisted mainly of β-O-4' linkages combined with small amounts of β-β', β-5', and α-O-4/β-O-4 linkages. Antioxidant activities of the lignins obtained were tested by the 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid), and ferric reducing activity power methods. It was found that alkali-extracted lignins obtained during the initial extraction process had higher antioxidant activities than alkaline ethanol-extracted lignins obtained during the second extraction process. PMID:24927101

  3. Archaeological Activity Report: Post-Review Discoveries Within 45BN431 at Solid Waste Site 128-F-2

    SciTech Connect

    T. E. Marceau; J. J. Sharpe

    2006-12-21

    During monitoring of remedial activities at Solid Waste Site 128-F-2 on August 19, 2005, a concentration of mussel shell was discovered in the west wall of a trench in the northen section of the waste site.

  4. A Unique Chitinase with Dual Active Sites and Triple Substrate Binding Sites from the Hyperthermophilic Archaeon Pyrococcus kodakaraensis KOD1

    PubMed Central

    Tanaka, Takeshi; Fujiwara, Shinsuke; Nishikori, Shingo; Fukui, Toshiaki; Takagi, Masahiro; Imanaka, Tadayuki

    1999-01-01

    We have found that the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 produces an extracellular chitinase. The gene encoding the chitinase (chiA) was cloned and sequenced. The chiA gene was found to be composed of 3,645 nucleotides, encoding a protein (1,215 amino acids) with a molecular mass of 134,259 Da, which is the largest among known chitinases. Sequence analysis indicates that ChiA is divided into two distinct regions with respective active sites. The N-terminal and C-terminal regions show sequence similarity with chitinase A1 from Bacillus circulans WL-12 and chitinase from Streptomyces erythraeus (ATCC 11635), respectively. Furthermore, ChiA possesses unique chitin binding domains (CBDs) (CBD1, CBD2, and CBD3) which show sequence similarity with cellulose binding domains of various cellulases. CBD1 was classified into the group of family V type cellulose binding domains. In contrast, CBD2 and CBD3 were classified into that of the family II type. chiA was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for chitinase activity were found to be 85°C and 5.0, respectively. Results of thin-layer chromatography analysis and activity measurements with fluorescent substrates suggest that the enzyme is an endo-type enzyme which produces a chitobiose as a major end product. Various deletion mutants were constructed, and analyses of their enzyme characteristics revealed that both the N-terminal and C-terminal halves are independently functional as chitinases and that CBDs play an important role in insoluble chitin binding and hydrolysis. Deletion mutants which contain the C-terminal half showed higher thermostability than did N-terminal-half mutants and wild-type ChiA. PMID:10583986

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

    PubMed Central

    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

  6. Strategies and Activities for Using Local Communities as Environmental Education Sites.

    ERIC Educational Resources Information Center

    Roth, Charles E.; Lockwood, Linda G.

    Presented are over 100 environmental education activities which use the local community for a learning site and resource. These lessons are grouped under seven topical headings: (1) biological neighbors, (2) physical environs, (3) built environs, (4) social environs, (5) understanding ourselves, (6) influencing change, and (7) improvement and…

  7. 77 FR 5830 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-06

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

  8. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2010 CFR

    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...

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

    ERIC Educational Resources Information Center

    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…

  10. Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

    PubMed Central

    Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

    2009-01-01

    Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

  11. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 1 2014-07-01 2014-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  12. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 1 2011-07-01 2011-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  13. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 1 2013-07-01 2013-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  14. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 1 2012-07-01 2012-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  15. The Thumbs Up Ecology Curriculum: A Fun Group of School Site Activities for Sixth Graders.

    ERIC Educational Resources Information Center

    Smith, John; And Others

    This guide is a collection of "fun" school site activities for sixth graders. Some of the topics covered are: animals, trees, energy and lifestyle, land use and you, energy conservation, and car-pooling. Each section offers both introductory information about the topic as well as questions to ponder such as what, so what, now what, and another way…

  16. IN VIVO ACTIVITY OF RHOPALOSIPHUM PADI VIRUS INTERNAL RIBOSOME ENTRY SITES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The RNA genome of Rhopalosiphum padi virus (RhPV), like other members of the Dicistroviridae, contains two open reading frames that are preceded by internal ribosome entry sites (IRESs). To compare the activities of the two RhPV IRESs in insect cells, a system was established for the in vivo transc...

  17. Cyclic silicate active site and stereochemical match for apatite nucleation on pseudowollastonite bioceramic-bone interfaces.

    PubMed

    Sahai, Nita; Anseau, Michel

    2005-10-01

    Hydroxyapatite (Ca5(PO4)3(OH)) forms on pseudowollastonite (psW) (alpha-CaSiO3) in vitro in simulated body fluid, human parotid saliva and cell-culture medium, and in vivo in implanted rat tibias. We used crystallographic constraints with ab initio molecular orbital calculations to identify the active site and reaction mechanism for heterogeneous nucleation of the earliest calcium phosphate oligomer/phase. The active site is the planar, cyclic, silicate trimer (Si3O9) on the (001) face of psW. The trimer has three silanol groups (>SiOH) arranged at 60 degrees from each other, providing a stereochemical match for O atoms bonded to Ca2+ on the (001) face of hydroxyapatite. Calcium phosphate nucleation is modeled in steps as hydrolysis of surface Ca-O bonds with leaching of Ca2+ into solution, protonation of the surface Si-O groups to form silanols, calcium sorption as an inner-sphere surface complex and, attachment of HPO4(2-). Our model explains the experimental solution and high resolution transmission electron microscopy data for epitaxial hydroxyapatite growth on psW in vitro and in vivo. We propose that the cyclic silicate trimer is the universal active site for heterogeneous, stereochemically promoted nucleation on silicate-based bioactive ceramics. A critical active site-density and a point of zero charge of the bioceramic less than physiological pH are required for bioactivity. PMID:15949543

  18. Bithionol Potently Inhibits Human Soluble Adenylyl Cyclase through Binding to the Allosteric Activator Site.

    PubMed

    Kleinboelting, Silke; Ramos-Espiritu, Lavoisier; Buck, Hannes; Colis, Laureen; van den Heuvel, Joop; Glickman, J Fraser; Levin, Lonny R; Buck, Jochen; Steegborn, Clemens

    2016-04-29

    The signaling molecule cAMP regulates functions ranging from bacterial transcription to mammalian memory. In mammals, cAMP is synthesized by nine transmembrane adenylyl cyclases (ACs) and one soluble AC (sAC). Despite similarities in their catalytic domains, these ACs differ in regulation. Transmembrane ACs respond to G proteins, whereas sAC is uniquely activated by bicarbonate. Via bicarbonate regulation, sAC acts as a physiological sensor for pH/bicarbonate/CO2, and it has been implicated as a therapeutic target, e.g. for diabetes, glaucoma, and a male contraceptive. Here we identify the bisphenols bithionol and hexachlorophene as potent, sAC-specific inhibitors. Inhibition appears mostly non-competitive with the substrate ATP, indicating that they act via an allosteric site. To analyze the interaction details, we solved a crystal structure of an sAC·bithionol complex. The structure reveals that the compounds are selective for sAC because they bind to the sAC-specific, allosteric binding site for the physiological activator bicarbonate. Structural comparison of the bithionol complex with apo-sAC and other sAC·ligand complexes along with mutagenesis experiments reveals an allosteric mechanism of inhibition; the compound induces rearrangements of substrate binding residues and of Arg(176), a trigger between the active site and allosteric site. Our results thus provide 1) novel insights into the communication between allosteric regulatory and active sites, 2) a novel mechanism for sAC inhibition, and 3) pharmacological compounds targeting this allosteric site and utilizing this mode of inhibition. These studies provide support for the future development of sAC-modulating drugs. PMID:26961873

  19. The Luminous Polycyclic Aromatic Hydrocarbon Emission Features: Applications to High Redshift Galaxies and Active Galactic Nuclei

    NASA Astrophysics Data System (ADS)

    Shipley, Heath; Papovich, Casey

    2015-08-01

    We provide a new robust star-formation rate (SFR) calibration using the luminosity from polycyclic aromatic hydrogen (PAH) molecules. The PAH features emit strongly in the mid-infrared (mid-IR; 3-19μm), mitigating dust extinction, and they are very luminous, containing 5-10% of the total IR luminosity in galaxies. We derive the calibration of the PAH luminosity as a SFR indicator using a sample of 105 star-forming galaxies covering a range of total IR luminosity, LIR = L(8-1000μm) = 109 - 1012 L⊙ and redshift 0 < z < 0.6. The PAH luminosity correlates linearly with the SFR as measured by the dust-corrected Hα luminosity (using the sum of the Hα and rest-frame 24μm luminosity from Kennicutt et al. 2009), with tight scatter of ~0.15 dex, comparable to the scatter in the dust-corrected Hα SFRs and Paα SFRs. We show this relation is sensitive to galaxy metallicity, where the PAH luminosity of galaxies with Z < 0.7 Z⊙ departs from the linear SFR relationship but in a behaved manor. We derive for this a correction to galaxies below solar metallicity. As a case study for observations with JWST, we apply the PAH SFR calibration to a sample of lensed galaxies at 1 < z < 3 with Spitzer Infrared Spectrograph (IRS) data, and we demonstrate the utility of PAHs to derive SFRs as accurate as those available from any other indicator. This new SFR indicator will be useful for probing the peak of the SFR density of the universe (1 < z < 3) and for studying the coevolution of star-formation and supermassive blackhole accretion contemporaneously in a galaxy.

  20. Let's Meet Famous Artists. A Creative Art Activity Book. A Teacher's Guide Featuring 19 Famous Artists and Art Activities.

    ERIC Educational Resources Information Center

    Kinghorn, Harriet; And Others

    By studying the lives, important achievements, and works of famous artists, students may come to understand what those artists hoped to communicate to the world. By participating in art activities that are related to these artists' styles, students practice skills of observing, analyzing, and creating. This instructional guide provides…

  1. Classic to Contemporary: Famous Artists and Activities. A Teacher's Guide Featuring 19 Famous Artists' Biographies and Enrichment/Research Activities.

    ERIC Educational Resources Information Center

    Kinghorn, Harriet; Lewis-Spicer, Lisa

    By studying the lives, important achievements, and works of famous artists, students may come to understand what those artists hoped to communicate to the world. By participating in art activities that are related to these artists' styles, students practice skills of observing, analyzing, and creating. This instructional guide provides…

  2. Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents.

    PubMed

    Stepankova, Veronika; Khabiri, Morteza; Brezovsky, Jan; Pavelka, Anton