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Sample records for active site comparison

  1. 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…

  2. 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

  3. Comparison of the active-site design of molybdenum oxo-transfer enzymes by quantum mechanical calculations.

    PubMed

    Li, Jilai; Ryde, Ulf

    2014-11-17

    There are three families of mononuclear molybdenum enzymes that catalyze oxygen atom transfer (OAT) reactions, named after a typical example from each family, viz., dimethyl sulfoxide reductase (DMSOR), sulfite oxidase (SO), and xanthine oxidase (XO). These families differ in the construction of their active sites, with two molybdopterin groups in the DMSOR family, two oxy groups in the SO family, and a sulfido group in the XO family. We have employed density functional theory calculations on cluster models of the active sites to understand the selection of molybdenum ligands in the three enzyme families. Our calculations show that the DMSOR active site has a much stronger oxidative power than the other two sites, owing to the extra molybdopterin ligand. However, the active sites do not seem to have been constructed to make the OAT reaction as exergonic as possible, but instead to keep the reaction free energy close to zero (to avoid excessive loss of energy), thereby making the reoxidation (SO and XO) or rereduction of the active sites (DMSOR) after the OAT reaction facile. We also show that active-site models of the three enzyme families can all catalyze the reduction of DMSO and that the DMSOR model does not give the lowest activation barrier. Likewise, all three models can catalyze the oxidation of sulfite, provided that the Coulombic repulsion between the substrate and the enzyme model can be overcome, but for this harder reaction, the SO model gives the lowest activation barrier, although the differences are not large. However, only the XO model can catalyze the oxidation of xanthine, owing to its sulfido ligand.

  4. Subway construction activity influence on polycyclic aromatic hydrocarbons in fine particles: Comparison with a background mountainous site

    NASA Astrophysics Data System (ADS)

    Kong, Shaofei; Li, Xuxu; Li, Qi; Yin, Yan; Li, Li; Chen, Kui; Liu, Dantong; Yuan, Liang; Pang, Xiaobing

    2015-07-01

    Intensive construction activities worsened the surrounding atmospheric environment in China. Eighteen polycyclic aromatic hydrocarbons (PAHs) in fine particles (PM2.5) were collected at a subway construction site (SC) of Nanjing and compared with a regional background mountainous site (BM) to examine the influence of anthropogenic activities on concentrations, sources and health risks of PAHs. Average PAH concentrations at SC were higher than BM at a factor of about 5.9. All PAH species at SC were higher than BM, with the SC/BM ratios ranging from 1.3 (NaP) to 10.3 (BaP). PAH profiles differed for the two sites. The SC site had higher mass fractions of PAHs from coal combustion and vehicle emission, while the BM site held higher mass percentages of PAHs from long-range transported wood combustion and industrial activities. Lower temperature at BM may lead to the higher mass percentages of low ring PAHs. Coal combustion, traffic emissions and biomass burning were the common sources for PAHs at both SC and BM. Construction workers were exposed to higher BaPeq concentrations, nearly ten times of the background site and their lifetime cancer risk reached to 0.6 per 1,000,000 exposed worker, owing to the influence of coal combustion, vehicle emission and industrial activities at the surroundings of SC.

  5. Petrophysical characterization of first ever drilled core samples from an active CO2 storage site, the German Ketzin Pilot Site - Comparison with long term experiments

    NASA Astrophysics Data System (ADS)

    Zemke, Kornelia; Liebscher, Axel

    2014-05-01

    Petrophysical properties like porosity and permeability are key parameters for a safe long-term storage of CO2 but also for the injection operation itself. These parameters may change during and/or after the CO2 injection due to geochemical reactions in the reservoir system that are triggered by the injected CO2. Here we present petrophysical data of first ever drilled cores from a newly drilled well at the active CO2 storage site - the Ketzin pilot site in the Federal State of Brandenburg, Germany. By comparison with pre-injection baseline data from core samples recovered prior to injection, the new samples provide the unique opportunity to evaluate the impact of CO2 on pore size related properties of reservoir and cap rocks at a real injection site under in-situ reservoir conditions. After injection of 61 000 tons CO2, an additional well was drilled and new rock cores were recovered. In total 100 core samples from the reservoir and the overlaying caprock were investigated by NMR relaxation. Permeability of 20 core samples was estimated by nitrogen and porosity by helium pycnometry. The determined data are comparable between pre-injection and post-injection core samples. The lower part of the reservoir sandstone is unaffected by the injected CO2. The upper part of the reservoir sandstone shows consistently slightly lower NMR porosity and permeability values in the post-injection samples when compared to the pre-injection data. This upper sandstone part is above the fluid level and CO2 present as a free gas phase and a possible residual gas saturation of the cores distorted the NMR results. The potash-containing drilling fluid can also influence these results: NMR investigation of twin samples from inner and outer parts of the cores show a reduced fraction of larger pores for the outer core samples together with lower porosities and T2 times. The drill mud penetration depth can be controlled by the added fluorescent tracer. Due to the heterogeneous character of the

  6. Oxygen reduction and evolution at single-metal active sites: Comparison between functionalized graphitic materials and protoporphyrins

    NASA Astrophysics Data System (ADS)

    Calle-Vallejo, F.; Martínez, J. I.; García-Lastra, J. M.; Abad, E.; Koper, M. T. M.

    2013-01-01

    A worldwide spread of clean technologies such as low-temperature fuel cells and electrolyzers depends strictly on their technical reliability and economic affordability. Currently, both conditions are hardly fulfilled mainly due to the same reason: the oxygen electrode, which has large overpotentials and is made of precious materials. A possible solution is the use of non-noble electrocatalysts with single-metal active sites. Here, on the basis of DFT calculations of adsorbed intermediates and a thermodynamic analysis, we compare the oxygen reduction (ORR) and evolution (OER) activities of functionalized graphitic materials and gas-phase porphyrins with late transition metals. We find that both kinds of materials follow approximately the same activity trends, and active sites with transition metals from groups 7 to 9 may be good ORR and OER electrocatalysts. However, spin analyses show more flexibility in the possible oxidation states of the metal atoms in solid electrocatalysts, while in porphyrins they must be + 2. These observations reveal that the catalytic activity of these materials is mainly due to nearest-neighbor interactions. Based on this, we propose that this class of electrocatalysts may be improved by careful selections of the support and the ligand properties close to the active sites and/or the ramifications near them, so that charge is transferred back and forth during adsorption and selective hydrogen bonds are formed.

  7. Active layer warming and deepening at Thule, Greenland during past decade: a comparison of a Polar Desert and a Polar Semi-desert site

    NASA Astrophysics Data System (ADS)

    Sletten, R. S.; Hagedorn, B.; Hallet, B.; Burnham, J. L.

    2013-12-01

    Microclimate and soil temperature/moisture monitoring at a Polar Desert and a Polar Semi-Desert site at Thule, Greenland reveals recent warming trends and distinct differences between these two sites. Approximately 5 C of warming occurred at the Polar Desert site from 2004 to 2009, while the Polar Semi-desert site warmed only about 3.5 C. Since 2009, the temperatures remain higher but stable. This warming trend is also reflected in the active layer, both in warming of the soil and in deepening of the active layer. Trends in soil temperatures and active layer depth are controlled substantially by the snow cover. For the Polar Semi-desert site, soil temperatures are measured both under natural snow accumulation conditions and under enhanced snow accumulation where a snow fence was installed. The insulating effect of the snow cover is effective and results in significantly warmer winter soils, as has been shown in other artificial snow cover studies. The warmer winter soils may allow greater respiration during the transition seasons thereby releasing more of the accumulated soil carbon in these sites than that we have previously documented. The trends in temperature and moisture may in part be controlled by the higher wind velocities at the Polar Desert site. These conditions are likely to be dominant controls on the ecosystem, along with differences in lithology which affects the cryoturbation of the soils. This in-depth microclimate study, along with our studies of the soils and soil water chemistry provides a detailed comparison of the adjacent ecosystems and may allow us to better predict how slight changes in climatic conditions may influence these systems and their subsequent feedback on carbon cycling.

  8. Modeling Active Layer Depth Over Permafrost for the Arctic Drainage Basin and the Comparison to Measurements at CALM Field Sites

    NASA Astrophysics Data System (ADS)

    Oelke, C.; Zhang, T.; Serreze, M.; Armstrong, R.

    2002-12-01

    A finite difference model for one-dimensional heat conduction with phase change is applied to investigate soil freezing and thawing processes over the Arctic drainage basin. Calculations are performed on the 25~km~x~25~km resolution NSIDC EASE-Grid. NCEP re-analyzed sigma-0.995 surface temperature with a topography correction, and SSM/I-derived weekly snow height are used as forcing parameters. The importance of using an annual cycle of snow density for different snow classes is emphasized. Soil bulk density and the percentages of silt/clay and sand/gravel are from the SoilData System of the International Geosphere Biosphere Programme. In addition, we parameterize a spatially and vertically variable peat layer and modify soil bulk density and thermal conductivity accordingly. Climatological soil moisture content is from the Permafrost/Water Balance Model (P/WBM) at the University of New Hampshire. The model domain is divided into 3~layers with distinct thermal properties of frozen and thawed soil, respectively. Calculations are performed on 54~model nodes ranging from a thickness of 10~cm near the surface to 1~m at 15~m depth. Initial temperatures are chosen according to the grid cell's IPA permafrost classification on EASE grid. Active layer depths, simulated for the summers of 1999 and 2000, compare well to maximal thaw depths measured at about 60 Circumarctic Active Layer Monitoring Network (CALM) field sites. A remaining RMS-error between modeled and measured values is attributed mainly to scale discrepancies (100~m~x~100~m vs. 25~km~x~25~km) based on differences in the fields of air temperature, snow height, and soil bulk density. For the whole pan-Arctic land mass and the time period 1980 through 2001, this study shows the regionally highly variable active layer depth, frozen ground depth, lengths of freezing and thawing periods, and the day of year when the maxima are reached.

  9. A Comparison of Vanadate to a 2'-5' Linkage at the Active Site of a Small Ribozyme Suggests a Role for Water in Transition-State Stabilization

    SciTech Connect

    Torelli, A.T.; Krucinska, J.; Wedekind, J.E.

    2009-06-04

    The potential for water to participate in RNA catalyzed reactions has been the topic of several recent studies. Here, we report crystals of a minimal, hinged hairpin ribozyme in complex with the transition-state analog vanadate at 2.05 A resolution. Waters are present in the active site and are discussed in light of existing views of catalytic strategies employed by the hairpin ribozyme. A second structure harboring a 2',5'-phosphodiester linkage at the site of cleavage was also solved at 2.35 A resolution and corroborates the assignment of active site waters in the structure containing vanadate. A comparison of the two structures reveals that the 2',5' structure adopts a conformation that resembles the reaction intermediate in terms of (1) the positioning of its nonbridging oxygens and (2) the covalent attachment of the 2'-O nucleophile with the scissile G+1 phosphorus. The 2',5'-linked structure was then overlaid with scissile bonds of other small ribozymes including the glmS metabolite-sensing riboswitch and the hammerhead ribozyme, and suggests the potential of the 2',5' linkage to elicit a reaction-intermediate conformation without the need to form metalloenzyme complexes. The hairpin ribozyme structures presented here also suggest how water molecules bound at each of the nonbridging oxygens of G+1 may electrostatically stabilize the transition state in a manner that supplements nucleobase functional groups. Such coordination has not been reported for small ribozymes, but is consistent with the structures of protein enzymes. Overall, this work establishes significant parallels between the RNA and protein enzyme worlds.

  10. Worldwide site comparison for submillimetre astronomy

    NASA Astrophysics Data System (ADS)

    Tremblin, P.; Schneider, N.; Minier, V.; Durand, G. Al.; Urban, J.

    2012-12-01

    Aims: The most important limitation for ground-based submillimetre (submm) astronomy is the broad-band absorption of the total water vapour in the atmosphere above an observation site, often expressed as the precipitable water vapour (PWV). A long-term statistic on the PWV is thus mandatory to characterize the quality of an existing or potential site for observational submm-astronomy. In this study we present a three-year statistic (2008-2010) of the PWV for ground-based telescope sites all around the world and for stratospheric altitudes relevant for SOFIA (Stratospheric Observatory for Far-Infrared Astronomy). The submm-transmission is calculated for typical PWVs using an atmospheric model. Methods: We used data from IASI (Infrared Atmospheric Sounding Interferometer) on the Metop-A satellite to retrieve water vapour profiles for each site (11 in total, comprising Antarctica, Chile, Mauna Kea, Greenland, Tibet). The use of a single instrument to make the comparison provides unbiased data with a common calibration method. The profiles are integrated above the mountain/stratospheric altitude to get an estimation of the PWV. We then applied the atmospheric model MOLIERE (Microwave Observation and LIne Estimation and REtrieval) to compute the corresponding atmospheric absorption for wavelengths between 150 μm and 3 mm. Results: We present the absolute PWV values for each site sorted by year and time percentage. The PWV corresponding to the first decile (10%) and the quartiles (25%, 50%, 75%) are calculated and transmission curves between 150 μm and 3 mm for these values are shown. The Antarctic and South-American sites present very good conditions for submillimetre astronomy. The 350 μm and 450 μm atmospheric windows are open all year long, whereas the 200 μm atmospheric window opens reasonably for 25% of the time in Antarctica and the extremely high-altitude sites in Chile. Potential interesting new facilities are Macon in Argentina and Summit in Greenland

  11. 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.

  12. Comparison Analysis among Large Amount of SNS Sites

    NASA Astrophysics Data System (ADS)

    Toriumi, Fujio; Yamamoto, Hitoshi; Suwa, Hirohiko; Okada, Isamu; Izumi, Kiyoshi; Hashimoto, Yasuhiro

    In recent years, application of Social Networking Services (SNS) and Blogs are growing as new communication tools on the Internet. Several large-scale SNS sites are prospering; meanwhile, many sites with relatively small scale are offering services. Such small-scale SNSs realize small-group isolated type of communication while neither mixi nor MySpace can do that. However, the studies on SNS are almost about particular large-scale SNSs and cannot analyze whether their results apply for general features or for special characteristics on the SNSs. From the point of view of comparison analysis on SNS, comparison with just several types of those cannot reach a statistically significant level. We analyze many SNS sites with the aim of classifying them by using some approaches. Our paper classifies 50,000 sites for small-scale SNSs and gives their features from the points of network structure, patterns of communication, and growth rate of SNS. The result of analysis for network structure shows that many SNS sites have small-world attribute with short path lengths and high coefficients of their cluster. Distribution of degrees of the SNS sites is close to power law. This result indicates the small-scale SNS sites raise the percentage of users with many friends than mixi. According to the analysis of their coefficients of assortativity, those SNS sites have negative values of assortativity, and that means users with high degree tend to connect users with small degree. Next, we analyze the patterns of user communication. A friend network of SNS is explicit while users' communication behaviors are defined as an implicit network. What kind of relationships do these networks have? To address this question, we obtain some characteristics of users' communication structure and activation patterns of users on the SNS sites. By using new indexes, friend aggregation rate and friend coverage rate, we show that SNS sites with high value of friend coverage rate activate diary postings

  13. Comparison of Savannah River Site`s meteorological databases

    SciTech Connect

    Weber, A.H.

    1993-07-01

    A five-year meteorological database from the 61-meter, H-Area tower for the period 1987--1991 was compared to an earlier database for the period 1982--1986. The amount of invalid data for the newer 87--91 database was one third that for the earlier database. The data recovery percentage for the last four years of the 87-91 database was well above 90%. Considerable effort was necessary to fill in for missing data periods for the newer database for the H-Area tower. Therefore, additional databases that have been prepared for the remaining SRS meteorological towers have had missing and erroneous data flagged, but not replaced. The F-Area tower`s database was used for cross-comparison purposes because of its proximity to H Area. The primary purpose of this report is to compare the H-Tower databases for 82-86 and 87-91. Statistical methods enable the use of probability statements to be made concerning the hypothesis of no differences between the distributions of the two time periods, assuming each database is a random sample from its respective distribution. This assumption is required for the statistical tests to be valid. A number of statistical comparisons can be made between the two data sets, even though the 82-86 database exist only as distributions of frequency and mean speed.

  14. 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.

  15. Comparison of the kinetics of S-S bond, secondary structure, and active site formation during refolding of reduced denatured hen egg white lysozyme.

    PubMed Central

    Roux, P.; Ruoppolo, M.; Chaffotte, A. F.; Goldberg, M. E.

    1999-01-01

    To investigate the role of some tertiary interactions, the disulfide bonds, in the early stages of refolding of hen lysozyme, we report the kinetics of reoxidation of denatured and reduced lysozyme under the same refolding conditions as those previously used to investigate the kinetics of regain of its circular dichroism (CD), fluorescence, and activity. At different stages of the refolding, the oxidation of the protein was blocked by alkylation of the free cysteines with iodoacetamide and the various oxidation states present in the samples were identified by electrospray-mass spectrometry. Thus, it was possible to monitor the appearance and/or disappearance of the species with 0 to 4 disulfide bonds. Using a simulation program, these kinetics were compared with those of regain of far-UV CD, fluorescence, and enzymatic activity and were discussed in terms of a refined model for the refolding of reduced hen egg white lysozyme. PMID:10631992

  16. Catalysis: Elusive active site in focus

    NASA Astrophysics Data System (ADS)

    Labinger, Jay A.

    2016-08-01

    The identification of the active site of an iron-containing catalyst raises hopes of designing practically useful catalysts for the room-temperature conversion of methane to methanol, a potential fuel for vehicles. See Letter p.317

  17. 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

  18. Identification and determination of trapping parameters as key site parameters for CO2 storage for the active CO2 storage site in Ketzin (Germany) - Comparison of different experimental approaches and analysis of field data

    NASA Astrophysics Data System (ADS)

    Zemke, Kornelia; Liebscher, Axel

    2015-04-01

    Petrophysical properties like porosity and permeability are key parameters for a safe long-term storage of CO2 but also for the injection operation itself. The accurate quantification of residual trapping is difficult, but very important for both storage containment security and storage capacity; it is also an important parameter for dynamic simulation. The German CO2 pilot storage in Ketzin is a Triassic saline aquifer with initial conditions of the target sandstone horizon of 33.5 ° C/6.1 MPa at 630 m. One injection and two observation wells were drilled in 2007 and nearly 200 m of core material was recovered for site characterization. From June 2008 to September 2013, slightly more than 67 kt food-grade CO2 has been injected and continuously monitored. A fourth observation well has been drilled after 61 kt injected CO2 in summer 2012 at only 25 m distance to the injection well and new core material was recovered that allow study CO2 induced changes in petrophysical properties. The observed only minor differences between pre-injection and post-injection petrophysical parameters of the heterogeneous formation have no severe consequences on reservoir and cap rock integrity or on the injection behavior. Residual brine saturation for the Ketzin reservoir core material was estimated by different methods. Brine-CO2 flooding experiments for two reservoir samples resulted in 36% and 55% residual brine saturation (Kiessling, 2011). Centrifuge capillary pressure measurements (pc = 0.22 MPa) yielded the smallest residual brine saturation values with ~20% for the lower part of the reservoir sandstone and ~28% for the upper part (Fleury, 2010). The method by Cerepi (2002), which calculates the residual mercury saturation after pressure release on the imbibition path as trapped porosity and the retracted mercury volume as free porosity, yielded unrealistic low free porosity values of only a few percent, because over 80% of the penetrated mercury remained in the samples after

  19. Cloud computing for protein-ligand binding site comparison.

    PubMed

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery. PMID:23762824

  20. Cloud computing for protein-ligand binding site comparison.

    PubMed

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  1. 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.

  2. Comparison of Candidate Sites for installation of Landfill facility at Ignalina NPP Site Using Fuzzy Logic Approach

    SciTech Connect

    Poskas, P.; Kilda, R.; Poskas, G.

    2008-07-01

    There is only one nuclear power plant in Lithuania - Ignalina NPP (Nuclear Power Plant). Two similar units with installed capacity of 1500 MW (each) were commissioned in 1983 and 1987 respectively. But the first Unit of Ignalina NPP was finally shutdown December 31, 2004, and second Unit is planned to be shutdown before 2010. Operational radioactive waste of different activities is generated at Ignalina NPP. After closure of INPP a waste from decommissioning should be managed also. According to Lithuanian regulatory requirements (1) the waste depending on the activity must be managed in different ways. In compliance with this Regulation very low-level radioactive waste (VLLW) could be disposed of in a Landfill facility. In such case very simple engineered barriers are required. A cap on the top of the repository is necessary from long-term safety point of view. Experience has shown that the effective and safe isolation of waste depends on the performance of the overall disposal system, which is formed by three major components: the site, the disposal facility and the waste form. The basic objective of the siting process is to select a suitable site for disposal and demonstrate that this site has characteristics which provide adequate isolation of radionuclides from the biosphere for desired periods of time. The methodology and results on evaluation and comparison of two candidate sites intended for construction of Landfill facility at Ignalina NPP site are presented in the paper. Criteria for comparison are based on the IAEA (International Atomic Energy Agency) recommendations (2). Modeling of the radionuclide releases has been performed using ISAM (Improving of Safety Assessment Methodologies for Near Surface Disposal facilities) methodology (3). For generalization of the information and elaboration of the recommendations Fuzzy Logic approach was used (4). (authors)

  3. Comparison of alternative cogeneration power systems for three industrial sites

    SciTech Connect

    Harper, A.D.

    1983-01-01

    Three alternative on-site cogeneration power systems were evaluated against technical and economic criteria for three industrial sites. Technical factors included plant sizing to meet process thermal loads, fuel utilization, power output, siting consideration, fuel savings, etc. Economic factors included capital cost, return on equity, and ownership/financing options among others. Each cogeneration plant was evaluated by comparison with the current separate generation scheme. The technologies considered were conventional coal-fired, steam topping cycles; coal-fired, atmospheric fluidized bed/closed-cycle gas turbines; and coal-fired, atmospheric fluidized bed/open cycle gas turbines. These approaches were optimized for three sites: an agricultural chemical plant, a brewery, and a kraft paper mill. The results showed that the closed cycle gas turbines yielded the best economics, primarily due to a lower initial cost. The open cycle gas turbine, when combined with a steam bottoming cycle, resulted in larger power outputs than would be realized in the closed cycle or steam turbine cases. None of the plants studied matched the plant electrical load while following the thermal load.

  4. Active Sites Environmental Monitoring Program: Action levels

    SciTech Connect

    Ashwood, J.S.; Ashwood, T.L.

    1991-10-01

    The Active Sites Environmental Monitoring Program (ASEMP) was established at Oak Ridge National Laboratory to provide for early leak detection and to monitor performance of the active low-level waste disposal facilities in Solid Waste Storage Area (SWSA) 6 and the transuranic waste storage areas in SWSA 5 North. Early leak detection is accomplished by sampling runoff, groundwater, and perched water in burial trenches. Sample results are compared to action levels that represent background contamination by naturally occurring and fallout-derived radionuclides. 15 refs., 3 figs., 12 tabs.

  5. Atmospheric stability comparisons at shore and inland sites

    SciTech Connect

    Brown, R.M.; SethuRaman, S.; Nagle, C.

    1980-01-01

    The values needed to predict diffusion and resulting concentrations from sources near coastlines is limited by lack of experimental data at coastal locations. The results obtained in this study are directed to assist in those calculations. Wind measurements made at the coastal site and some comparisons with the measurements made at an inland location, both on Long Island, are presented. Continuous wind speed and direction measurements have been made at the Tiana Beach site since 1975. Hourly averaged wind direction, speed and gustiness types were obtained from these records, and processed by computer techniques. Simultaneous data from the inland location were also obtained. The data were grouped with a digital computer according to gustiness types, wind-speed categories and 10/sup 0/ wind direction increments from 0 to 360 degrees. The initial analysis was to separate the various gustiness types by the number of occurrences per month at each location. These data were used to determine the ratio of each type to the total monthly hours at each place. The ratios at each location were compared to give some understanding of the differences of total gustiness between the coastal and inland site.

  6. Characterization of active sites in zeolite catalysts

    SciTech Connect

    Eckert, J.; Bug, A.; Nicol, J.M.

    1997-11-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Atomic-level details of the interaction of adsorbed molecules with active sites in catalysts are urgently needed to facilitate development of more effective and/or environmentally benign catalysts. To this end the authors have carried out neutron scattering studies combined with theoretical calculations of the dynamics of small molecules inside the cavities of zeolite catalysts. The authors have developed the use of H{sub 2} as a probe of adsorption sites by observing the hindered rotations of the adsorbed H{sub 2} molecule, and they were able to show that an area near the four-rings is the most likely adsorption site for H{sub 2} in zeolite A while adsorption of H{sub 2} near cations located on six-ring sites decreases in strength as Ni {approximately} Co > Ca > Zn {approximately} Na. Vibrational and rotational motions of ethylene and cyclopropane adsorption complexes were used as a measure for zeolite-adsorbate interactions. Preliminary studies of the binding of water, ammonia, and methylamines were carried out in a number of related guest-host materials.

  7. Early Site Permit Demonstration Program, plant parameters envelopes: Comparison with ranges of values for four hypothetical sites. Volume 2

    SciTech Connect

    Not Available

    1992-09-01

    The purpose of this volume is to report the results of the comparison of the ALWR plan parameters envelope with values of site characteristics developed for our hypothetical sites that generally represent conditions encountered within the United States. This effort is not intended to identify or address the suitability of any existing site, site area, or region in the United States. Also included in this volume is Appendix F, SERCH Summaries Regarding Siting.

  8. Comparison of lactate sampling sites for rock climbing.

    PubMed

    Fryer, S; Draper, N; Dickson, T; Blackwell, G; Winter, D; Ellis, G

    2011-06-01

    Comparisons of capillary blood lactate concentrations pre and post climb have featured in the protocols of many rock climbing studies, with most researchers obtaining samples from the fingertip. The nature of rock climbing, however, places a comparatively high physiological loading on the foreaand fingertips. Indeed, the fingertips are continually required for gripping and this makes pre-climb sampling at this site problematic. The purpose of our study was to examine differences in capillary blood lactate concentrations from samples taken at the fingertip and first (big) toe in a rock climbing context. 10 participants (9 males and 1 female) completed climbing bouts at 3 different angles (91°, 100° and 110°). Capillary blood samples were taken simultaneously from the fingertip and first toe pre and post climb. A limit of agreement plot revealed all data points to be well within the upper and lower bounds of the 95% population confidence interval. Subsequent regression analysis revealed a strong relationship (R (2)=0.94, y=0.940x + 0.208) between fingertip and first toe capillary blood lactate concentrations. Findings from our study suggest that the toe offers a valid alternative site for capillary blood lactate concentration analysis in a rock climbing context.

  9. 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.

  10. Site characterization in densely fractured dolomite: Comparison of methods

    USGS Publications Warehouse

    Muldoon, M.; Bradbury, K.R.

    2005-01-01

    One of the challenges in characterizing fractured-rock aquifers is determining whether the equivalent porous medium approximation is valid at the problem scale. Detailed hydrogeologic characterization completed at a small study site in a densely fractured dolomite has yielded an extensive data set that was used to evaluate the utility of the continuum and discrete-fracture approaches to aquifer characterization. There are two near-vertical sets of fractures at the site; near-horizontal bedding-plane partings constitute a third fracture set. Eighteen boreholes, including five coreholes, were drilled to a depth of ???10.6 m. Borehole geophysical logs revealed several laterally extensive horizontal fractures and dissolution zones. Flowmeter and short-interval packer testing identified which of these features were hydraulically important. A monitoring system, consisting of short-interval piezometers and multilevel samplers, was designed to monitor four horizontal fractures and two dissolution zones. The resulting network consisted of >70 sampling points and allowed detailed monitoring of head distributions in three dimensions. Comparison of distributions of hydraulic head - and hydraulic conductivity determined by these two approaches suggests that even in a densely fractured-carbonate aquifer, a characterization approach using traditional long-interval monitoring wells is inadequate to characterize ground water movement for the purposes of regulatory monitoring or site remediation. In addition, traditional multiwell pumping tests yield an average or bulk hydraulic conductivity that is not adequate for predicting rapid ground water travel times through the fracture network, and the pumping test response does not appear to be an adequate tool for assessing whether the porous medium approximation is valid. Copyright ?? 2005 National Ground Water Association.

  11. Site characterization in densely fractured dolomite: comparison of methods.

    PubMed

    Muldoon, Maureen; Bradbury, Ken R

    2005-01-01

    One of the challenges in characterizing fractured-rock aquifers is determining whether the equivalent porous medium approximation is valid at the problem scale. Detailed hydrogeologic characterization completed at a small study site in a densely fractured dolomite has yielded an extensive data set that was used to evaluate the utility of the continuum and discrete-fracture approaches to aquifer characterization. There are two near-vertical sets of fractures at the site; near-horizontal bedding-plane partings constitute a third fracture set. Eighteen boreholes, including five coreholes, were drilled to a depth of approximately 10.6 m. Borehole geophysical logs revealed several laterally extensive horizontal fractures and dissolution zones. Flowmeter and short-interval packer testing identified which of these features were hydraulically important. A monitoring system, consisting of short-interval piezometers and multilevel samplers, was designed to monitor four horizontal fractures and two dissolution zones. The resulting network consisted of >70 sampling points and allowed detailed monitoring of head distributions in three dimensions. Comparison of distributions of hydraulic head and hydraulic conductivity determined by these two approaches suggests that even in a densely fractured-carbonate aquifer, a characterization approach using traditional long-interval monitoring wells is inadequate to characterize ground water movement for the purposes of regulatory monitoring or site remediation. In addition, traditional multiwell pumping tests yield an average or bulk hydraulic conductivity that is not adequate for predicting rapid ground water travel times through the fracture network, and the pumping test response does not appear to be an adequate tool for assessing whether the porous medium approximation is valid.

  12. 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.

  13. 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.

  14. Histidine at the active site of Neurospora tyrosinase.

    PubMed

    Pfiffner, E; Lerch, K

    1981-10-13

    The involvement of histidyl residues as potential ligands to the binuclear active-site copper of Neurospora tyrosinase was explored by dye-sensitized photooxidation. The enzymatic activity of the holoenzyme was shown to be unaffected by exposure to light in the presence of methylene blue; however, irradiation of the apoenzyme under the same conditions led to a progressive loss of its ability to be reactivated with Cu2+. This photoinactivation was paralleled by a decrease in the histidine content whereas the number of histidyl residues in the holoenzyme remained constant. Copper measurements of photooxidized, reconstituted apoenzyme demonstrated the loss of binding of one copper atom per mole of enzyme as a consequence of photosensitized oxidation of three out of nine histidine residues. Their sequence positions were determined by a comparison of the relative yields of the histidine containing peptides of photooxidized holo- and apotyrosinases. The data obtained show the preferential modification of histidyl residues 188, 193, and 289 and suggest that they constitute metal ligands to one of the two active-site copper atoms. Substitution of copper by cobalt was found to afford complete protection of the histidyl residues from being modified by dye-sensitized photooxidation. PMID:6458322

  15. Control of active sites in flocculation: Concept of equivalent active sites''

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Flocculation and dispersion of solids are strong functions of the amount and conformation of the adsorbed polymer. Regions of dispersion and flocculation of solids with particular polymer molecules may be deduced from saturation adsorption data. The concept of equivalent active sites'' is proposed to explain flocculation and dispersion behavior irrespective of the amount or conformation of the adsorbed polymer. The concept has been further extended to study the selective flocculation process.

  16. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases.

    PubMed

    Indra, A K; Warot, X; Brocard, J; Bornert, J M; Xiao, J H; Chambon, P; Metzger, D

    1999-11-15

    Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ER(T) or Cre-ER(T2) under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T). PMID:10536138

  17. Mass and number size distributions of particulate matter components: comparison of an industrial site and an urban background site.

    PubMed

    Taiwo, Adewale M; Beddows, David C S; Shi, Zongbo; Harrison, Roy M

    2014-03-15

    Size-resolved composition of particulate matter (PM) sampled in the industrial town of Port Talbot (PT), UK was determined in comparison to a typical urban background site in Birmingham (EROS). A Micro-Orifice Uniform Deposit Impactor (MOUDI) sampler was deployed for two separate sampling campaigns with the addition of a Grimm optical spectrometer at the PT site. MOUDI samples were analysed for water-soluble anions (Cl(-), NO₃(-) and SO₄(2-)) and cations (Na(+), NH4(+), K(+), Mg(2+) and Ca(2+)) and trace metals (Al, V, Cr, Mn, Fe, Cu, Zn, Sb, Ba and Pb). The PM mass distribution showed a predominance of fine particle (PM₂.₅) mass at EROS whereas the PT samples were dominated by the coarse fraction (PM₂.₅₋₁₀). SO₄(2-), Cl(-), NH4(+), Na(+), NO₃(-), and Ca(2+) were the predominant ionic species at both sites while Al and Fe were the metals with highest concentrations at both sites. Mean concentrations of Cl(-), Na(+), K(+), Ca(2+), Mg(2+), Cr, Mn, Fe and Zn were higher at PT than EROS due to industrial and marine influences. The contribution of regional pollution by sulphate, ammonium and nitrate was greater at EROS relative to PT. The traffic signatures of Cu, Sb, Ba and Pb were particularly prominent at EROS. Overall, PM at EROS was dominated by secondary aerosol and traffic-related particles while PT was heavily influenced by industrial activities and marine aerosol. Profound influences of wind direction are seen in the 72-hour data, especially in relation to the PT local sources. Measurements of particle number in 14 separate size bins plotted as a function of wind direction and speed are highly indicative of contributing sources, with local traffic dominant below 0.5 μm, steelworks emissions from 0.5 to 15 μm, and marine aerosol above 15 μm. PMID:24419284

  18. Comparison of potential power production at on- and offshore sites

    NASA Astrophysics Data System (ADS)

    Pryor, S. C.; Barthelmie, R. J.

    2001-10-01

    This article presents analyses of the potential power production from turbines located in the near-shore and offshore environment relative to an onshore location, using half-hourly average wind speed data from four sites in the Danish wind monitoring network. These measurement sites are located in a relatively high wind speed environment, and data from these sites indicate a high degree of spatial coherence. For these sites and representative turbine specifications (rated power 1.3-2 W) the fraction of time with power output in excess of 500 kW is twice as high for the offshore location as for the land site. Also, the fraction of time with negligible power production (defined as <100 kW output from the turbines described herein) is less than 20% for the offshore site and twice as high at the land-based location. Capacity factors are higher for coastal sites than for the land site, and the annual capacity factor for the offshore location is twice that of the land site. Potential power output at the offshore site exhibits approximately the same seasonal variation as at the land site but little diurnal variation.

  19. 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.

  20. 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?

  1. 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.

  2. 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.

  3. Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site*S

    PubMed Central

    Maresso, Anthony W.; Wu, Ruiying; Kern, Justin W.; Zhang, Rongguang; Janik, Dorota; Missiakas, Dominique M.; Duban, Mark-Eugene; Joachimiak, Andrzej; Schneewind, Olaf

    2011-01-01

    Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (β-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via β-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery. PMID:17545669

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. Identification of inhibitors against the potential ligandable sites in the active cholera toxin.

    PubMed

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

    The active cholera toxin responsible for the massive loss of water and ions in cholera patients via its ADP ribosylation activity is a heterodimer of the A1 subunit of the bacterial holotoxin and the human cytosolic ARF6 (ADP Ribosylation Factor 6). The active toxin is a potential target for the design of inhibitors against cholera. In this study we identified the potential ligandable sites of the active cholera toxin which can serve as binding sites for drug-like molecules. By employing an energy-based approach to identify ligand binding sites, and comparison with the results of computational solvent mapping, we identified two potential ligandable sites in the active toxin which can be targeted during structure-based drug design against cholera. Based on the probe affinities of the identified ligandable regions, docking-based virtual screening was employed to identify probable inhibitors against these sites. Several indole-based alkaloids and phosphates showed strong interactions to the important residues of the ligandable region at the A1 active site. On the other hand, 26 top scoring hits were identified against the ligandable region at the A1 ARF6 interface which showed strong hydrogen bonding interactions, including guanidines, phosphates, Leucopterin and Aristolochic acid VIa. This study has important implications in the application of hybrid structure-based and ligand-based methods against the identified ligandable sites using the identified inhibitors as reference ligands, for drug design against the active cholera toxin.

  9. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.

    PubMed Central

    Weaver, T.; Lees, M.; Banaszak, L.

    1997-01-01

    Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation. PMID:9098893

  10. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

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

  11. Comparison of computer codes and inputs used at DOE sites to model intrusion scenarios

    SciTech Connect

    Seitz, R.R.; Cook, J.R.; Wood, M.I.; Rittman, P.D.; Wood, D.E.; Napier, B.A.

    1994-09-01

    Scenarios and computer codes used to evaluate intrusion scenarios at different DOE sites are compared and discussed. The purpose of the comparison is to identify differences in the approaches and areas where approaches could be made more consistent without ignoring the need to consider legimate site-specific differences, The comparison is comprised of two steps: (1) benchmarking of CENII and PATHRAE (the two most commonly used codes at DOE sites), and (2) comparison of assumed values for selected input parameters from:scenarios used at the different sites. The results of the benchmarking and parameter comparisons identify fundamental differences in the default assumptions used in the computer codes, as well as differences in the approaches used at the different sites. GENII and PATHRAE are applied to a variety of sites within DOE and other regulatory environments, differences in default assumptions identified in these comparisons are discussed for users to consider when they apply the codes. Furthermore, differences in assumptions made at the different sites disposing of low-level radioactive wastes within Department of Energy are identified to provide an example of how the Performance Assessment Task Team is working to ensure consistent interpretation of performance assessment results.

  12. A COMPARISON OF APPROACHES TO PRIORITIZING SITES FOR RIPARIAN RESTORATION

    EPA Science Inventory

    This study compares the results of Olson and Harris (1997) and Russell et al.(1997)in their work to prioritize sites for riparian restoration in the San Luis Rey River watershed. Olson and Harris defined reaches of the mainstem and evaluated the relative potential for restoration...

  13. Classification and comparison of ligand-binding sites derived from grid-mapped knowledge-based potentials.

    PubMed

    Hoppe, Christian; Steinbeck, Christoph; Wohlfahrt, Gerd

    2006-03-01

    We describe the application of knowledge-based potentials implemented in the MOE program to compare the ligand-binding sites of several proteins. The binding probabilities for a polar and a hydrophobic probe are calculated on a grid to allow easy comparison of binding sites of superimposed related proteins. The method is fast and simple enough to simultaneously use structural information of multiple proteins of a target family. The method can be used to rapidly cluster proteins into subfamilies according to the similarity of hydrophobic and polar fields of their ligand-binding sites. Regions of the binding site which are common within a protein family can be identified and analysed for the design of family-targeted libraries or those which differ for improvement of ligand selectivity. The field-based hierarchical clustering is demonstrated for three protein families: the ligand-binding domains of nuclear receptors, the ATP-binding sites of protein kinases and the substrate binding sites of proteases. More detailed comparisons are presented for serine proteases of the chymotrypsin family, for the peroxisome proliferator-activated receptor subfamily of nuclear receptors and for progesterone and androgen receptor. The results are in good accordance with structure-based analysis and highlight important differences of the binding sites, which have been also described in the literature.

  14. Site comparison of selected aerosol samplers in the wood industry.

    PubMed

    Kauffer, Edmond; Wrobel, Richard; Görner, Peter; Rott, Christelle; Grzebyk, Michel; Simon, Xavier; Witschger, Olivier

    2010-03-01

    Several samplers (IOM, CIP 10-I v1, ACCU-CAP, and Button) were evaluated at various wood industry companies using the CALTOOL system. The results obtained show that compared to the CALTOOL mouth, which can be considered to be representative of the exposure of a person placed at the same location under the same experimental conditions, the concentrations measured by the IOM, CIP 10-I v1, and ACCU-CAP samplers are not significantly different (respectively, 1.12, 0.94, and 0.80 compared to 1.00), the Button sampler (0.86) being close to the ACCU-CAP sampler. Comparisons of dust concentrations measured using both a closed-face cassette (CFC) and one of the above samplers were also made. In all, 235 sampling pairs (sampler + CFC) taken at six companies provided us with a comparison of concentrations measured using IOM, CIP 10-I v1, ACCU-CAP, and Button samplers with concentrations measured using a CFC. All the studied samplers collected systematically more dust than the CFC (2.0 times more for the IOM sampler, 1.84 times more for the CIP 10-I v1 sampler, 1.68 times more for the ACCU-CAP sampler, and 1.46 times more for the Button sampler). The literature most frequently compares the IOM sampler with the CFC: published results generally show larger differences compared with the CFC than those found during our research. There are several explanations for this difference, one of which involves CFC orientation during sampling. It has been shown that concentrations measured using a CFC are dependent on its orientation. Different CFC positions from one sampling session to another are therefore likely to cause differences during CFC-IOM sampler comparisons.

  15. Characterization of the active site of chloroperoxidase using physical techniques

    SciTech Connect

    Hall, K.S.

    1986-01-01

    Chloroperoxidase (CPO) and Cytochrome P-450, two very different hemeproteins, have been shown to have similar active sites by several techniques. Recent work has demonstrated thiolate ligation from a cysteine residue to the iron in P-450. A major portion of this research has been devoted to obtaining direct evidence that CPO also has a thiolate 5th ligand from a cysteine residue. This information will provide the framework for a detailed analysis of the structure-function relationships between peroxidases, catalase and cytochrome P-450 hemeproteins. To determine whether the 5th ligand is a cysteine, methionine or a unique amino acid, specific isotope enrichment experiments were used. Preliminary /sup 1/H-NMR studies show that the carbon monoxide-CPO complex has a peak in the upfield region corresponding to alpha-protons of a thiolate amino acid. C. fumago was grown on 95% D/sub 2/O media with a small amount of /sup 1/H-cysteine added. Under these conditions C. fumago slows down the biosynthesis of cysteine by at least 50% and utilizes the exogenous cysteine in the media. GC-MS was able to show that the methylene protons next to the sulfur atom in cysteine are 80-90% protonated while these positions in methionine are approximately 73% deuterated. Comparison of the /sup 1/H-NMR spectra of CO-CPO and CO-CPO indicate the presence of a cysteine ligand in chloroperoxidase.

  16. Impact of Binding Site Comparisons on Medicinal Chemistry and Rational Molecular Design.

    PubMed

    Ehrt, Christiane; Brinkjost, Tobias; Koch, Oliver

    2016-05-12

    Modern rational drug design not only deals with the search for ligands binding to interesting and promising validated targets but also aims to identify the function and ligands of yet uncharacterized proteins having impact on different diseases. Additionally, it contributes to the design of inhibitors with distinct selectivity patterns and the prediction of possible off-target effects. The identification of similarities between binding sites of various proteins is a useful approach to cope with those challenges. The main scope of this perspective is to describe applications of different protein binding site comparison approaches to outline their applicability and impact on molecular design. The article deals with various substantial application domains and provides some outstanding examples to show how various binding site comparison methods can be applied to promote in silico drug design workflows. In addition, we will also briefly introduce the fundamental principles of different protein binding site comparison methods.

  17. Catalyst Activity Comparison of Alcohols over Zeolites

    SciTech Connect

    Ramasamy, Karthikeyan K.; Wang, Yong

    2013-01-01

    Alcohol transformation to transportation fuel range hydrocarbon on HZSM-5 (SiO2 / Al2O3 = 30) catalyst was studied at 360oC and 300psig. Product distributions and catalyst life were compared using methanol, ethanol, 1-propanol or 1-butanol as a feed. The catalyst life for 1-propanol and 1-butanol was more than double compared to that for methanol and ethanol. For all the alcohols studied, the product distributions (classified to paraffin, olefin, napthene, aromatic and naphthalene compounds) varied with time on stream (TOS). At 24 hours TOS, liquid product from 1-propanol and 1-butanol transformation primarily contains higher olefin compounds. The alcohol transformation process to higher hydrocarbon involves a complex set of reaction pathways such as dehydration, oligomerization, dehydrocyclization, and hydrogenation. Compared to ethylene generated from methanol and ethanol, oligomerization of propylene and butylene has a lower activation energy and can readily take place on weaker acidic sites. On the other hand, dehydrocyclization of propylene and butylene to form the cyclic compounds requires the sits with stronger acid strength. Combination of the above mentioned reasons are the primary reasons for olefin rich product generated in the later stage of the time on stream and for the extended catalyst life time for 1 propanol and 1 butanol compared to methanol and ethanol conversion over HZSM-5.

  18. The Sim-SEQ Project: Comparison of Selected Flow Models for the S-3 Site

    SciTech Connect

    Mukhopadhyay, Sumit; Doughty, Christine A.; Bacon, Diana H.; Li, Jun; Wei, Lingli; Yamamoto, Hajime; Gasda, Sarah E.; Hosseini, Seyyed; Nicot, Jean-Philippe; Birkholzer, Jens

    2015-05-23

    Sim-SEQ is an international initiative on model comparison for geologic carbon sequestration, with an objective to understand and, if possible, quantify model uncertainties. Model comparison efforts in Sim-SEQ are at present focusing on one specific field test site, hereafter referred to as the Sim-SEQ Study site (or S-3 site). Within Sim-SEQ, different modeling teams are developing conceptual models of CO2 injection at the S-3 site. In this paper, we select five flow models of the S-3 site and provide a qualitative comparison of their attributes and predictions. These models are based on five different simulators or modeling approaches: TOUGH2/EOS7C, STOMP-CO2e, MoReS, TOUGH2-MP/ECO2N, and VESA. In addition to model-to-model comparison, we perform a limited model-to-data comparison, and illustrate how model choices impact model predictions. We conclude the paper by making recommendations for model refinement that are likely to result in less uncertainty in model predictions.

  19. 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.

  20. A novel approach to predict active sites of enzyme molecules.

    PubMed

    Chou, Kuo-Chen; Cai, Yu-dong

    2004-04-01

    Enzymes are critical in many cellular signaling cascades. With many enzyme structures being solved, there is an increasing need to develop an automated method for identifying their active sites. However, given the atomic coordinates of an enzyme molecule, how can we predict its active site? This is a vitally important problem because the core of an enzyme molecule is its active site from the viewpoints of both pure scientific research and industrial application. In this article, a topological entity was introduced to characterize the enzymatic active site. Based on such a concept, the covariant discriminant algorithm was formulated for identifying the active site. As a paradigm, the serine hydrolase family was demonstrated. The overall success rate by jackknife test for a data set of 88 enzyme molecules was 99.92%, and that for a data set of 50 independent enzyme molecules was 99.91%. Meanwhile, it was shown through an example that the prediction algorithm can also be used to find any typographic error of a PDB file in annotating the constituent amino acids of catalytic triad and to suggest a possible correction. The very high success rates are due to the introduction of a covariance matrix in the prediction algorithm that makes allowance for taking into account the coupling effects among the key constituent atoms of active site. It is anticipated that the novel approach is quite promising and may become a useful high throughput tool in enzymology, proteomics, and structural bioinformatics. PMID:14997541

  1. Growth exponents in surface models with non-active sites

    NASA Astrophysics Data System (ADS)

    Santos, M.; Figueiredo, W.; Aarão Reis, F. D. A.

    2006-11-01

    In this work, we studied the role played by the inactive sites present on the substrate of a growing surface. In our model, one particle sticks at the surface if the site where it falls is an active site. However, we allow the deposited particle to diffuse along the surface in accordance with some mechanism previously defined. Using Monte Carlo simulations, and some analytical results, we have investigated the model in (1+1) and (2+1) dimensions considering different relaxation mechanisms. We show that the consideration of non-active sites is a crucial point in the model. In fact, we have seen that the saturation regime is not observed for any value of the density of inactive sites. Besides, the growth exponent β turns to be one, at long times, whatever the mechanism of diffusion we consider in one and two dimensions.

  2. 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

  3. TOSPAC calculations in support of the COVE 2A benchmarking activity; Yucca Mountain Site Characterization Project

    SciTech Connect

    Gauthier, J.H.; Zieman, N.B.; Miller, W.B.

    1991-10-01

    The purpose of the the Code Verification (COVE) 2A benchmarking activity is to assess the numerical accuracy of several computer programs for the Yucca Mountain Site Characterization Project of the Department of Energy. This paper presents a brief description of the computer program TOSPAC and a discussion of the calculational effort and results generated by TOSPAC for the COVE 2A problem set. The calculations were performed twice. The initial calculations provided preliminary results for comparison with the results from other COVE 2A participants. TOSPAC was modified in response to the comparison and the final calculations included a correction and several enhancements to improve efficiency. 8 refs.

  4. Comparison of Two Computer Algorithms To Identify Surgical Site Infections

    PubMed Central

    Apte, Mandar; Landers, Timothy; Furuya, Yoko; Hyman, Sandra

    2011-01-01

    Abstract Background Surgical site infections (SSIs), the second most common healthcare-associated infections, increase hospital stay and healthcare costs significantly. Traditional surveillance of SSIs is labor-intensive. Mandatory reporting and new non-payment policies for some SSIs increase the need for efficient and standardized surveillance methods. Computer algorithms using administrative, clinical, and laboratory data collected routinely have shown promise for complementing traditional surveillance. Methods Two computer algorithms were created to identify SSIs in inpatient admissions to an urban, academic tertiary-care hospital in 2007 using the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnosis codes (Rule A) and laboratory culture data (Rule B). We calculated the number of SSIs identified by each rule and both rules combined and the percent agreement between the rules. In a subset analysis, the results of the rules were compared with those of traditional surveillance in patients who had undergone coronary artery bypass graft surgery (CABG). Results Of the 28,956 index hospital admissions, 5,918 patients (20.4%) had at least one major surgical procedure. Among those and readmissions within 30 days, the ICD-9-CM-only rule identified 235 SSIs, the culture-only rule identified 287 SSIs; combined, the rules identified 426 SSIs, of which 96 were identified by both rules. Positive and negative agreement between the rules was 36.8% and 97.1%, respectively, with a kappa of 0.34 (95% confidence interval [CI] 0.27–0.41). In the subset analysis of patients who underwent CABG, of the 22 SSIs identified by traditional surveillance, Rule A identified 19 (86.4%) and Rule B identified 13 (59.1%) cases. Positive and negative agreement between Rules A and B within these “positive controls” was 81.3% and 50.0% with a kappa of 0.37 (95% CI 0.04–0.70). Conclusion Differences in the rates of SSI identified by computer

  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)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

  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. Mercury transfer from soil to olive trees. A comparison of three different contaminated sites.

    PubMed

    Higueras, Pablo L; Amorós, José Á; Esbrí, José Maria; Pérez-de-los-Reyes, Caridad; López-Berdonces, Miguel A; García-Navarro, Francisco J

    2016-04-01

    Mercury contents in soil and olive tree leaves have been studied in 69 plots around three different source areas of this element in Spain: Almadén (Ciudad Real), Flix (Tarragona) and Jódar (Jaén). Almadén was the world's largest cinnabar (HgS) mining district and was active until 2003, Flix is the oldest Spanish chlor-alkali plant (CAP) and has been active from 1898 to the present day and Jódar is a decommissioned CAP that was active for 14 years (1977-1991). Total mercury contents have been measured by high-frequency modulation atomic absorption spectrometry with Zeeman effect (ZAAS-HFM) in the soils and olive tree leaves from the three studied areas. The average soil contents range from 182 μg kg(-1) in Flix to 23,488 μg kg(-1) in Almadén, while the average leaf content ranges from 161 μg kg(-1) in Jódar to 1213 μg kg(-1) in Almadén. Despite the wide range of data, a relationship between soil-leaf contents has been identified: in Almadén and Jódar, multiplicative (bilogarithmic) models show significant correlations (R = 0.769 and R = 0.484, respectively). Significant correlations were not identified between soil and leaf contents in Flix. The continuous activity of the Flix CAP, which remains open today, can explain the different uptake patterns for mercury, which is mainly atmospheric in origin, in comparison to the other two sites, where activity ceased more than 10 years ago and only soil uptake patterns based on the Michaelis-Menten enzymatic model curve are observed.

  9. Mercury transfer from soil to olive trees. A comparison of three different contaminated sites.

    PubMed

    Higueras, Pablo L; Amorós, José Á; Esbrí, José Maria; Pérez-de-los-Reyes, Caridad; López-Berdonces, Miguel A; García-Navarro, Francisco J

    2016-04-01

    Mercury contents in soil and olive tree leaves have been studied in 69 plots around three different source areas of this element in Spain: Almadén (Ciudad Real), Flix (Tarragona) and Jódar (Jaén). Almadén was the world's largest cinnabar (HgS) mining district and was active until 2003, Flix is the oldest Spanish chlor-alkali plant (CAP) and has been active from 1898 to the present day and Jódar is a decommissioned CAP that was active for 14 years (1977-1991). Total mercury contents have been measured by high-frequency modulation atomic absorption spectrometry with Zeeman effect (ZAAS-HFM) in the soils and olive tree leaves from the three studied areas. The average soil contents range from 182 μg kg(-1) in Flix to 23,488 μg kg(-1) in Almadén, while the average leaf content ranges from 161 μg kg(-1) in Jódar to 1213 μg kg(-1) in Almadén. Despite the wide range of data, a relationship between soil-leaf contents has been identified: in Almadén and Jódar, multiplicative (bilogarithmic) models show significant correlations (R = 0.769 and R = 0.484, respectively). Significant correlations were not identified between soil and leaf contents in Flix. The continuous activity of the Flix CAP, which remains open today, can explain the different uptake patterns for mercury, which is mainly atmospheric in origin, in comparison to the other two sites, where activity ceased more than 10 years ago and only soil uptake patterns based on the Michaelis-Menten enzymatic model curve are observed. PMID:25801370

  10. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site

    PubMed Central

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R.; Phillips, Chris; Augustin, Martin A.; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-01-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5–inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented. PMID:27139631

  11. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site.

    PubMed

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R; Phillips, Chris; Augustin, Martin A; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-05-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.

  12. 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.

  13. 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.

  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.

  15. 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

  16. Studies on the active site of pig plasma amine oxidase.

    PubMed Central

    Collison, D; Knowles, P F; Mabbs, F E; Rius, F X; Singh, I; Dooley, D M; Cote, C E; McGuirl, M

    1989-01-01

    Amine oxidase from pig plasma (PPAO) has two bound Cu2+ ions and at least one pyrroloquinoline quinone (PQQ) moiety as cofactors. It is shown that recovery of activity by copper-depleted PPAO is linear with respect to added Cu2+ ions. Recovery of e.s.r. and optical spectral characteristics of active-site copper parallel the recovery of catalytic activity. These results are consistent with both Cu2+ ions contributing to catalysis. Further e.s.r. studies indicate that the two copper sites in PPAO, unlike those in amine oxidases from other sources, are chemically distinct. These comparative studies establish that non-identity of the Cu2+ ions in PPAO is not a requirement for amine oxidase activity. It is shown through the use of a new assay procedure that there are two molecules of PQQ bound per molecule of protein in PPAO; only the more reactive of these PQQ moieties is required for activity. PMID:2559715

  17. ConSite: web-based prediction of regulatory elements using cross-species comparison.

    PubMed

    Sandelin, Albin; Wasserman, Wyeth W; Lenhard, Boris

    2004-07-01

    ConSite is a user-friendly, web-based tool for finding cis-regulatory elements in genomic sequences. Predictions are based on the integration of binding site prediction generated with high-quality transcription factor models and cross-species comparison filtering (phylogenetic footprinting). By incorporating evolutionary constraints, selectivity is increased by an order of magnitude as compared to single-sequence analysis. ConSite offers several unique features, including an interactive expert system for retrieving orthologous regulatory sequences. Programming modules and biological databases that form the foundation of the ConSite service are freely available to the research community. ConSite is available at http:/www.phylofoot.org/consite.

  18. Computer simulation of the active site of human serum cholinesterase

    SciTech Connect

    Kefang Jiao; Song Li; Zhengzheng Lu

    1996-12-31

    The first 3D-structure of acetylchelinesterase from Torpedo California electric organ (T.AChE) was published by JL. Sussman in 1991. We have simulated 3D-structure of human serum cholinesterase (H.BuChE) and the active site of H.BuChE. It is discovered by experiment that the residue of H.BuChE is still active site after a part of H.BuChE is cut. For example, the part of 21KD + 20KD is active site of H.BuChE. The 20KD as it is. Studies on these peptides by Hemelogy indicate that two active peptides have same negative electrostatic potential maps diagram. These negative electrostatic areas attached by acetyl choline with positive electrostatic potency. We predict that 147...236 peptide of AChE could be active site because it was as 20KD as with negative electrostatic potential maps. We look forward to proving from other ones.

  19. Risk management study for the Hanford Site facilities: Risk reduction cost comparison for the retired Hanford Site facilities. Volume 4

    SciTech Connect

    Coles, G.A.; Egge, R.G.; Senger, E.; Shultz, M.W.; Taylor, W.E.

    1994-02-01

    This document provides a cost-comparison evaluation for implementing certain risk-reduction measures and their effect on the overall risk of the 100 and 200 Area retired, surplus facilities. The evaluation is based on conditions that existed at the time the risk evaluation team performed facility investigations, and does not acknowledge risk-reduction measures that occurred soon after risk identification. This evaluation is one part of an overall risk management study for these facilities. The retired facilities investigated for this evaluation are located in the 100 and 200 Areas of the 1450-km{sup 2} Hanford Site. The Hanford Site is a semiarid tract of land in southeastern Washington State. The nearest population center is Richland, Washington, (population 32,000) 30 km southeast of the 200 Area. This cost-comparison evaluation (1) determines relative costs for reducing risk to acceptable levels; (2) compares the cost of reducing risk using different risk-reduction options; and (3) compares the cost of reducing risks at different facilities. The result is an identification of the cost effective risk-reduction measures. Supporting information required to develop costs of the various risk-reduction options also is included.

  20. Multi-site Phosphorylation Regulates Bim Stability and Apoptotic Activity

    PubMed Central

    Hübner, Anette; Barrett, Tamera; Flavell, Richard A.; Davis, Roger J.

    2008-01-01

    The pro-apoptotic BH3-only protein Bim is established to be an important mediator of signaling pathways that induce cell death. Multi-site phosphorylation of Bim by several members of the MAP kinase group is implicated as a regulatory mechanism that controls the apoptotic activity of Bim. To test the role of Bim phosphorylation in vivo, we constructed mice with a series of mutant alleles that express phosphorylation-defective Bim proteins. We show that mutation of the phosphorylation site Thr-112 causes decreased binding of Bim to the anti-apoptotic protein Bcl2 and can increase cell survival. In contrast, mutation of the phosphorylation sites Ser-55, Ser-65, and Ser-73 can cause increased apoptosis because of reduced proteasomal degradation of Bim. Together, these data indicate that phosphorylation can regulate Bim by multiple mechanisms and that the phosphorylation of Bim on different sites can contribute to the sensitivity of cellular apoptotic responses. PMID:18498746

  1. D Reconstruction of AN Underwater Archaelogical Site: Comparison Between Low Cost Cameras

    NASA Astrophysics Data System (ADS)

    Capra, A.; Dubbini, M.; Bertacchini, E.; Castagnetti, C.; Mancini, F.

    2015-04-01

    The 3D reconstruction with a metric content of a submerged area, where objects and structures of archaeological interest are found, could play an important role in the research and study activities and even in the digitization of the cultural heritage. The reconstruction of 3D object, of interest for archaeologists, constitutes a starting point in the classification and description of object in digital format and for successive fruition by user after delivering through several media. The starting point is a metric evaluation of the site obtained with photogrammetric surveying and appropriate 3D restitution. The authors have been applying the underwater photogrammetric technique since several years using underwater digital cameras and, in this paper, digital low cost cameras (off-the-shelf). Results of tests made on submerged objects with three cameras are presented: Canon Power Shot G12, Intova Sport HD e GoPro HERO 2. The experimentation had the goal to evaluate the precision in self-calibration procedures, essential for multimedia underwater photogrammetry, and to analyze the quality of 3D restitution. Precisions obtained in the calibration and orientation procedures was assessed by using three cameras, and an homogeneous set control points. Data were processed with Agisoft Photoscan. Successively, 3D models were created and the comparison of the models derived from the use of different cameras was performed. Different potentialities of the used cameras are reported in the discussion section. The 3D restitution of objects and structures was integrated with sea bottom floor morphology in order to achieve a comprehensive description of the site. A possible methodology of survey and representation of submerged objects is therefore illustrated, considering an automatic and a semi-automatic approach.

  2. Rapid comparison of protein binding site surfaces with Property Encoded Shape Distributions (PESD)

    PubMed Central

    Das, Sourav; Kokardekar, Arshad

    2009-01-01

    Patterns in shape and property distributions on the surface of binding sites are often conserved across functional proteins without significant conservation of the underlying amino-acid residues. To explore similarities of these sites from the viewpoint of a ligand, a sequence and fold-independent method was created to rapidly and accurately compare binding sites of proteins represented by property-mapped triangulated Gauss-Connolly surfaces. Within this paradigm, signatures for each binding site surface are produced by calculating their property-encoded shape distributions (PESD), a measure of the probability that a particular property will be at a specific distance to another on the molecular surface. Similarity between the signatures can then be treated as a measure of similarity between binding sites. As postulated, the PESD method rapidly detected high levels of similarity in binding site surface characteristics even in cases where there was very low similarity at the sequence level. In a screening experiment involving each member of the PDBBind 2005 dataset as a query against the rest of the set, PESD was able to retrieve a binding site with identical E.C. (Enzyme Commission) numbers as the top match in 79.5% of cases. The ability of the method in detecting similarity in binding sites with low sequence conservations were compared with state-of-the-art binding site comparison methods. PMID:19919089

  3. 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

  4. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  5. 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.

  6. A Comparison of two Brunhes Chron Geomagnetic Excursions Recorded by Neighbouring North Atlantic Sites (ODP Sites 1062 and 1063)

    NASA Astrophysics Data System (ADS)

    Bourne, M.; Mac Niocaill, C.; Knudsen, M. F.; Thomas, A. L.; Henderson, G. M.

    2012-04-01

    core-mantle system. The long duration of fully reversed directions at the two sites is somewhat longer than that typically assumed for excursions and appears to suggest that there may be a degree of stability associated with the two excursional events. We will present a comparison of the geomagnetic field behaviour of the two excursions as recorded at these two sites.

  7. 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

  8. Conformational Transitions in Human AP Endonuclease 1 and Its Active Site Mutant during Abasic Site Repair†

    PubMed Central

    Kanazhevskaya, Lyubov Yu.; Koval, Vladimir V.; Zharkov, Dmitry O.; Strauss, Phyllis R.; Fedorova, Olga S.

    2010-01-01

    AP endonuclease 1 (APE 1) is a crucial enzyme of the base excision repair pathway (BER) in human cells. APE1 recognizes apurinic/apyrimidinic (AP) sites and makes a nick in the phosphodiester backbone 5′ to them. The conformational dynamics and presteady-state kinetics of wild-type APE1 and its active site mutant, Y171F-P173L-N174K, have been studied. To observe conformational transitions occurring in the APE1 molecule during the catalytic cycle, we detected intrinsic tryptophan fluorescence of the enzyme under single turnover conditions. DNA duplexes containing a natural AP site, its tetrahydrofuran analogue, or a 2′-deoxyguanosine residue in the same position were used as specific substrates or ligands. The stopped-flow experiments have revealed high flexibility of the APE1 molecule and the complexity of the catalytic process. The fluorescent traces indicate that wild-type APE1 undergoes at least four conformational transitions during the processing of abasic sites in DNA. In contrast, nonspecific interactions of APE1 with undamaged DNA can be described by a two-step kinetic scheme. Rate and equilibrium constants were extracted from the stopped-flow and fluorescence titration data for all substrates, ligands, and products. A replacement of three residues at the enzymatic active site including the replacement of tyrosine 171 with phenylalanine in the enzyme active site resulted in a 2 × 104-fold decrease in the reaction rate and reduced binding affinity. Our data indicate the important role of conformational changes in APE1 for substrate recognition and catalysis. PMID:20575528

  9. 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

  10. N-methyl-D-aspartate recognition site ligands modulate activity at the coupled glycine recognition site.

    PubMed

    Hood, W F; Compton, R P; Monahan, J B

    1990-03-01

    In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine-insensitive [3H]glycine binding are altered in the presence of N-methyl-D-aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L-glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 microM vs. 0.129 microM). Similar decreases in affinity are observed for the glycine site agonists, D-serine and 1-aminocyclopropane-1-carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1-hydroxy-3-amino-2-pyrrolidone and 1-aminocyclobutane-1-carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L-glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.

  11. 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

  12. Control of active sites in selective flocculation: I -- Mathematical model

    SciTech Connect

    Behl, S.; Moudgil, B.M.; Prakash, T.S. . Dept. of Materials Science and Engineering)

    1993-12-01

    Heteroflocculation has been determined to be another major reason for loss in selectivity for flocculation process. In a mathematical model developed earlier, conditions for controlling heteroflocculation were discussed. Blocking active sites to control selective adsorption of a flocculant oil a desirable solid surface is discussed. It has been demonstrated that the lower molecular weight fraction of a flocculant which is incapable of flocculating the particles is an efficient site blocking agent. The major application of selective flocculation has been in mineral processing but many potential uses exist in biological and other colloidal systems. These include purification of ceramic powders, separating hazardous solids from chemical waste, and removal of deleterious components from paper pulp.

  13. Comparison of Metalloproteinase Protein and Activity Profiling

    PubMed Central

    Giricz, Orsi; Lauer, Janelle L.; Fields, Gregg B.

    2010-01-01

    Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The present study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer and one normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media, both in amount and in variety. TIMP-1 was produced in all cell lines. MMP activity assays with three different FRET substrates were then utilized to determine if protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was only confirmed in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays. PMID:20920458

  14. The site of activation of factor X by cancer procoagulant.

    PubMed

    Gordon, S G; Mourad, A M

    1991-12-01

    Cancer procoagulant (CP) is a cysteine proteinase found in a variety of malignant cells and tissues and in human amnion-chorion tissue. It initiates coagulation by activating factor X. However, the amino acid sequence of the substrate protein that determines the cleavage site of cysteine proteinases is different from that of the serine proteinases that normally activate factor X, such as factor IXa, VIIa and Russell's Viper Venom (RVV). Therefore, it was of interest to determine the site of cleavage of human factor X by CP. Purified CP was incubated with purified factor X and the reaction mixture was electrophoresed on a 10% Tris-tricine SDS-PAGE gel. The proteins were electroeluted on to a polyvinylidene difluoride (PVDF) membrane, and stained with Coomassie blue. The heavy chain of activated factor X was cut out of the PVDF membrane and sequenced with an Applied Biosystems 477A with on-line HPLC. The primary cleavage sequence was Asp-Ala-Ala-Asp-Leu-Asp-Pro-; two other secondary sequences Ser-Ile-Thr-Trp-Lys-Pro- and Glu-Asn-Pro-Phe-Asp-Leu were found. The penultimate amino acid on the carbonyl side of the hydrolysed amide bond plays a critical role for the recognition of the cleavage site of cysteine proteinases. These data indicate that the penultimate amino acid for the primary cleavage site of factor X by CP is proline-20 and for the secondary sites, proline-13 and proline-28. This is in contrast to arginine-52 that determines the specificity of the cleavage by normal serine proteinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. 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.

  16. Functional constituents of the active site of human neutrophil collagenase.

    PubMed

    Mookhtiar, K A; Wang, F; Van Wart, H E

    1986-05-01

    A series of chemical modification reactions has been carried out to identify functional constituents of the active site of human neutrophil collagenase. The enzyme is reversibly inhibited by the transition metal chelating agent 1,10-phenanthroline, and inhibition is fully reversed by zinc. Removal of weakly bound metal ions by gel filtration inactivates collagenase, and activity is fully restored on immediate readdition of calcium. The enzyme is unaffected by reagents that modify serine, cysteine, and arginine residues. However, reaction with the carboxyl reagents cyclohexylmorpholinocarbodiimide and Woodward's Reagent K lowers the activity of the enzyme substantially. Acetylimidazole inactivates the enzyme, but activity is completely restored on addition of hydroxylamine. The enzyme is also inactivated by tetranitromethane, indicating that it contains an essential tyrosine residue. Acylation of collagenase with diethyl pyrocarbonate, diketene, acetic anhydride, or trinitrobenzenesulfonate inactivates the enzyme, and activity is not restored on addition of hydroxylamine, indicating the presence of an essential lysine residue.

  17. 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.

  18. 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

  19. Active sites in char gasification: Final technical report

    SciTech Connect

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

    1987-09-01

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

  20. Active Sites Environmental Monitoring Program: FY 1991 report

    SciTech Connect

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

    1992-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from April 1991 through September 1991. The ASEMP was established in 1989 by Solid Waste Operations (SWO) and the Environmental Sciences Division, both of Oak Ridge National Laboratory, 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. A new set of action levels was developed on the basis of a statistical analysis of background contamination. These new action levels have been used to evaluate results in this report. Results of ASEMP monitoring continue to demonstrate that no LLW (except [sup 3]H) is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II, which began in early FY 1991, was >90% complete at the end of September 1991. Results of sampling of groundwater and surface waters is presented.

  1. Inhibition and active-site modelling of prolidase.

    PubMed

    King, G F; Crossley, M J; Kuchel, P W

    1989-03-15

    Consideration of the active-site model of prolidase led us to examine azetidine, pyrrolidine and piperidine substrate analogs as potential in vivo inhibitors of the enzyme. One of these, N-benzyloxycarbonyl-L-proline, was shown to be a potent competitive inhibitor of porcine kidney prolidase (Ki = 90 microM); its rapid protein-mediated permeation of human and sheep erythrocytes suggests that it may be effective in vivo. The higher homolog, N-benzyloxycarbonyl-L-pipecolic acid, was also a potent inhibitor of the enzyme while the antihypertensive drugs, captopril and enalaprilat, were shown to have mild and no inhibitory effects, respectively. Analysis of inhibitor action and consideration of X-ray crystallographic data of relevant Mn2+ complexes allowed the active-site model of prolidase to be further refined; a new model is presented in which the substrate acts as a bidentate ligand towards the active-site manganous ion. Various aspects of the new model help to explain why Mn2+ has been 'chosen' by the enzyme in preference to other biologically available metal ions. PMID:2924773

  2. Hidden relationships between metalloproteins unveiled by structural comparison of their metal sites.

    PubMed

    Valasatava, Yana; Andreini, Claudia; Rosato, Antonio

    2015-03-30

    Metalloproteins account for a substantial fraction of all proteins. They incorporate metal atoms, which are required for their structure and/or function. Here we describe a new computational protocol to systematically compare and classify metal-binding sites on the basis of their structural similarity. These sites are extracted from the MetalPDB database of minimal functional sites (MFSs) in metal-binding biological macromolecules. Structural similarity is measured by the scoring function of the available MetalS2 program. Hierarchical clustering was used to organize MFSs into clusters, for each of which a representative MFS was identified. The comparison of all representative MFSs provided a thorough structure-based classification of the sites analyzed. As examples, the application of the proposed computational protocol to all heme-binding proteins and zinc-binding proteins of known structure highlighted the existence of structural subtypes, validated known evolutionary links and shed new light on the occurrence of similar sites in systems at different evolutionary distances. The present approach thus makes available an innovative viewpoint on metalloproteins, where the functionally crucial metal sites effectively lead the discovery of structural and functional relationships in a largely protein-independent manner.

  3. Hidden relationships between metalloproteins unveiled by structural comparison of their metal sites

    NASA Astrophysics Data System (ADS)

    Valasatava, Yana; Andreini, Claudia; Rosato, Antonio

    2015-03-01

    Metalloproteins account for a substantial fraction of all proteins. They incorporate metal atoms, which are required for their structure and/or function. Here we describe a new computational protocol to systematically compare and classify metal-binding sites on the basis of their structural similarity. These sites are extracted from the MetalPDB database of minimal functional sites (MFSs) in metal-binding biological macromolecules. Structural similarity is measured by the scoring function of the available MetalS2 program. Hierarchical clustering was used to organize MFSs into clusters, for each of which a representative MFS was identified. The comparison of all representative MFSs provided a thorough structure-based classification of the sites analyzed. As examples, the application of the proposed computational protocol to all heme-binding proteins and zinc-binding proteins of known structure highlighted the existence of structural subtypes, validated known evolutionary links and shed new light on the occurrence of similar sites in systems at different evolutionary distances. The present approach thus makes available an innovative viewpoint on metalloproteins, where the functionally crucial metal sites effectively lead the discovery of structural and functional relationships in a largely protein-independent manner.

  4. Role of arginine-304 in the diphosphate-triggered active site closure mechanism of trichodiene synthase.

    PubMed

    Vedula, L Sangeetha; Cane, David E; Christianson, David W

    2005-09-27

    The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PP(i)) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PP(i) bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg2+(3)-PP(i), in which R304 donates hydrogen bonds to PP(i) and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP(i) in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP(i) and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg2+(3)-PP(i) binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP(i) leaving group. PMID:16171386

  5. Role of Arginine-304 in the Diphosphate-Triggered Active Site Closure Mechanism of Trichodiene Synthase

    SciTech Connect

    Vedula,L.; Cane, D.; Christianson, D.

    2005-01-01

    The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PPi) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PPi bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg{sup 2+}{sub 3-}PP{sub i}, in which R304 donates hydrogen bonds to PP{sub i} and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP{sub i} in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP{sub i} and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg{sup 2+}{sub 3-}PP{sub i} binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP{sub i} leaving group.

  6. Active-Site Structure of Class IV Adenylyl Cyclase and Transphyletic Mechanism

    SciTech Connect

    D Gallagher; S Kim; H Robinson; P Reddy

    2011-12-31

    Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3',5'-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV) - two with substrate analogs and one with product. Mn{sup 2+} binds to all three phosphates, and to Glu12 and Glu136. Electropositive residues Lys14, Arg63, Lys76, Lys111, and Arg113 also form hydrogen bonds to phosphates. The conformation of the analogs is suitable for in-line nucleophilic attack by the ribose O3' on {alpha}-phosphate (distance {approx} 4 {angstrom}). In the product complex, a second Mn ion is observed to be coordinated to both ribose 2' oxygen and ribose 3' oxygen. Observation of both metal sites, together with kinetic measurements, provides strong support for a two-cation mechanism. Eleven active-site mutants were also made and kinetically characterized. These findings and comparisons with class II and class III enzymes enable a detailed transphyletic analysis of the AC mechanism. Consistent with its lack of coordination to purine, Y. pestis AC-IV cyclizes both ATP and GTP. As in other classes of AC, the ribose is loosely bound, and as in class III, no base appears to ionize the O3' nucleophile. Different syn/anti conformations suggest that the mechanism involves a conformational transition, and further evidence suggests a role for ribosyl pseudorotation. With resolutions of 1.6-1.7 {angstrom}, these are the most detailed active-site ligand complexes for any class of this ubiquitous signaling enzyme.

  7. Active-Site Structure of Class IV Adenylyl Cyclase and Transphyletic Mechanism

    SciTech Connect

    Gallagher, D.T.; Robinson, H.; Kim, S.-K.; Reddy, P. T.

    2011-01-21

    Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3',5'-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV)-two with substrate analogs and one with product. Mn{sup 2+} binds to all three phosphates, and to Glu12 and Glu136. Electropositive residues Lys14, Arg63, Lys76, Lys111, and Arg113 also form hydrogen bonds to phosphates. The conformation of the analogs is suitable for in-line nucleophilic attack by the ribose O3' on {alpha}-phosphate (distance {approx} 4 {angstrom}). In the product complex, a second Mn ion is observed to be coordinated to both ribose 2' oxygen and ribose 3' oxygen. Observation of both metal sites, together with kinetic measurements, provides strong support for a two-cation mechanism. Eleven active-site mutants were also made and kinetically characterized. These findings and comparisons with class II and class III enzymes enable a detailed transphyletic analysis of the AC mechanism. Consistent with its lack of coordination to purine, Y. pestis AC-IV cyclizes both ATP and GTP. As in other classes of AC, the ribose is loosely bound, and as in class III, no base appears to ionize the O3' nucleophile. Different syn/anti conformations suggest that the mechanism involves a conformational transition, and further evidence suggests a role for ribosyl pseudorotation. With resolutions of 1.6-1.7 {angstrom}, these are the most detailed active-site ligand complexes for any class of this ubiquitous signaling enzyme.

  8. Druggability analysis and classification of protein tyrosine phosphatase active sites

    PubMed Central

    Ghattas, Mohammad A; Raslan, Noor; Sadeq, Asil; Al Sorkhy, Mohammad; Atatreh, Noor

    2016-01-01

    Protein tyrosine phosphatases (PTP) play important roles in the pathogenesis of many diseases. The fact that no PTP inhibitors have reached the market so far has raised many questions about their druggability. In this study, the active sites of 17 PTPs were characterized and assessed for its ability to bind drug-like molecules. Consequently, PTPs were classified according to their druggability scores into four main categories. Only four members showed intermediate to very druggable pocket; interestingly, the rest of them exhibited poor druggability. Particularly focusing on PTP1B, we also demonstrated the influence of several factors on the druggability of PTP active site. For instance, the open conformation showed better druggability than the closed conformation, while the tight-bound water molecules appeared to have minimal effect on the PTP1B druggability. Finally, the allosteric site of PTP1B was found to exhibit superior druggability compared to the catalytic pocket. This analysis can prove useful in the discovery of new PTP inhibitors by assisting researchers in predicting hit rates from high throughput or virtual screening and saving unnecessary cost, time, and efforts via prioritizing PTP targets according to their predicted druggability. PMID:27757011

  9. Comparison of optimal irrigation scheduling and groundwater recharge at representative sites in the North China Plain

    NASA Astrophysics Data System (ADS)

    Ma, Ying

    2014-05-01

    The North China Plain (NCP) is an important food production area in China, facing an increasing water shortage and overexploitation of groundwater. It is critical to optimize the irrigation scheduling and accurately estimate groundwater recharge for saving water and increasing crop water use efficiency. However, the water cycle and crop responses to irrigation are quite various in different areas, because of the spatial variation of climatic, soil, water table and other management practices in the NCP. In this study, three representative sites (LC site in the piedmont plain, TZ site in the northern alluvial and lacustrine plain, YC site in the southern alluvial and lacustrine plain) were selected to compare the optimal irrigation scheduling and corresponding groundwater recharge under different hydrological years for winter wheat-summer maize double cropping system. At each site, a physically based agro-hydrological model (SWAP) was calibrated using field data of soil moisture. Then, scenarios under different irrigation time and amount were simulated. Results showed that the optimal irrigation scheduling and corresponding groundwater recharge were significant different between the three representative sites. The mean water table depth at the LC (33.0 m), YC (10.3 m), and TZ site (2.5 m) caused great different time lags of infiltrated water and groundwater contribution to evapotranspiration. Then, the most irrigation amount was required for the TZ site but the least requirement for the YC site at each hydrologic year. As most clay contents in the deep soils at the LC site increased tortuosity and limited water movement, which resulted in lower rates of recharge compared to more sandy soils at the other two sites. Averagely, using the optimal irrigation scheduling could save 2.04×109 m3 irrigation water and reduce about 84.3% groundwater over-exploitation in winter wheat growth period in the NCP. Therefore, comparison of the simulation results among the three

  10. 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.

  11. Comparison of marine gas hydrates in sediments of an active and passive continental margin

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

    Two sites of the Deep Sea Drilling Project in contrasting geologic settings provide a basis for comparison of the geochemical conditions associated with marine gas hydrates in continental margin sediments. Site 533 is located at 3191 m water depth on a spit-like extension of the continental rise on a passive margin in the Atlantic Ocean. Site 568, at 2031 m water depth, is in upper slope sediment of an active accretionary margin in the Pacific Ocean. Both sites are characterized by high rates of sedimentation, and the organic carbon contents of these sediments generally exceed 0.5%. Anomalous seismic reflections that transgress sedimentary structures and parallel the seafloor, suggested the presence of gas hydrates at both sites, and, during coring, small samples of gas hydrate were recovered at subbottom depths of 238m (Site 533) and 404 m (Site 568). The principal gaseous components of the gas hydrates wer methane, ethane, and CO2. Residual methane in sediments at both sites usually exceeded 10 mll-1 of wet sediment. Carbon isotopic compositions of methane, CO2, and ??CO2 followed parallel trends with depth, suggesting that methane formed mainly as a result of biological reduction of oxidized carbon. Salinity of pore waters decreased with depth, a likely result of gas hydrate formation. These geochemical characteristics define some of the conditions associated with the occurrence of gas hydrates formed by in situ processes in continental margin sediments. ?? 1984.

  12. Monitoring air quality with lichens: a comparison between mapping in forest sites and in open areas.

    PubMed

    Policnik, Helena; Simoncic, Primoz; Batic, Franc

    2008-01-01

    Four different methods of epiphytic lichen mapping were used for the assessment of air quality in the region under the influence of the Sostanj Thermal Power Plant (Salek Valley, Slovenia). Three methods were based on the presence of different lichen species (VDI, EU and ICP-Forest), the fourth on a frequency and coverage assessment of different growth forms of epiphytic lichens, e.g. crustose, foliose and fruticose (SI). A comparison of the results from the assessment of air quality between forest sites (ICP-Forest, SI) and open areas (VDI, EU and SI), obtained by the different methods of epiphytic lichen mapping, is presented in the contribution. Data showed that lichen species richness is worse in forest sites in comparison with open areas. From the data obtained it can be concluded that epiphytic lichen mapping in open areas is a better method for the assessment of air pollution in a given area than mapping in forest sites. The species-based methods in open areas are more powerful and useful for air quality assessment in polluted research areas than the SI and ICP-Forest methods.

  13. The pepsin residue glycine-76 contributes to active-site loop flexibility and participates in catalysis.

    PubMed Central

    Okoniewska, M; Tanaka, T; Yada, R Y

    2000-01-01

    Glycine residues are known to contribute to conformational flexibility of polypeptide chains, and have been found to contribute to flexibility of some loops associated with enzymic catalysis. A comparison of porcine pepsin in zymogen, mature and inhibited forms revealed that a loop (a flap), consisting of residues 71--80, located near the active site changed its position upon substrate binding. The loop residue, glycine-76, has been implicated in the catalytic process and thought to participate in a hydrogen-bond network aligning the substrate. This study investigated the role of glycine-76 using site-directed mutagenesis. Three mutants, G76A, G76V and G76S, were constructed to increase conformational restriction of a polypeptide chain. In addition, the serine mutant introduced a hydrogen-bonding potential at position 76 similar to that observed in human renin. All the mutants, regardless of amino acid size and polarity, had lower catalytic efficiency and activated more slowly than the wild-type enzyme. The slower activation process was associated directly with altered proteolytic activity. Consequently, it was proposed that a proteolytic cleavage represents a limiting step of the activation process. Lower catalytic efficiency of the mutants was explained as a decrease in the flap flexibility and, therefore, a different pattern of hydrogen bonds responsible for substrate alignment and flap conformation. The results demonstrated that flap flexibility is essential for efficient catalytic and activation processes. PMID:10861225

  14. Electrostatic fields in the active sites of lysozymes.

    PubMed

    Sun, D P; Liao, D I; Remington, S J

    1989-07-01

    Considerable experimental evidence is in support of several aspects of the mechanism that has been proposed for the catalytic activity of lysozyme. However, the enzymatically catalyzed hydrolysis of polysaccharides proceeds over 5 orders of magnitude faster than that of model compounds that mimic the configuration of the substrate in the active site of the enzyme. Although several possible explanations for this rate enhancement have been discussed elsewhere, a definitive mechanism has not emerged. Here we report striking results obtained by classical electrodynamics, which suggest that bond breakage and the consequent separation of charge in lysozyme is promoted by a large electrostatic field across the active site cleft, produced in part by a very asymmetric distribution of charged residues on the enzyme surface. Lysozymes unrelated in amino acid sequence have similar distributions of charged residues and electric fields. The results reported here suggest that the electrostatic component of the rate enhancement is greater than 9 kcal.mol-1. Thus, electrostatic interactions may play a more important role in the enzymatic mechanism than has generally been appreciated.

  15. Interlaboratory comparison on 137Cs activity concentration in fume dust

    NASA Astrophysics Data System (ADS)

    Tzika, Faidra; Hult, Mikael; Burda, Oleksiy; Arnold, Dirk; Sibbens, Goedele; Caro Marroyo, Belén; Gómez-Mancebo, Maria Belén; Peyrés, Virginia; Moser, Hannah; Ferreux, Laurent; Šolc, Jaroslav; Dryák, Pavel; Fazio, Aldo; Luca, Aurelian; Vodenik, Branko; Reis, Mario; Tyminski, Zbigniew; Klemola, Seppo

    2015-11-01

    A comparison was conducted, between 11 European National Metrology Institutes and EC-JRC, on measurement of 137Cs activity concentration in fume dust. As test material an activity standard produced from real contaminated fume dust was used. The standard material consisted of 13 cylindrical samples of compressed fume dust. The material contained 137Cs and 60Co of reference activity concentrations of (9.72±0.10) Bq/g and (0.450±0.018) Bq/g, respectively, for the reference date of 1 June 2013, determined using the comparison results. The organization and results of the intercomparison, as well as the process of obtaining reliable reference values are presented.

  16. Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

    PubMed

    Cane, D E; Shim, J H; Xue, Q; Fitzsimons, B C; Hohn, T M

    1995-02-28

    Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7873527

  17. 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

  18. Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

    PubMed Central

    Jakubík, J; Bacáková, L; Lisá, V; el-Fakahany, E E; Tucek, S

    1996-01-01

    Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation. PMID:8710935

  19. Comparison of charge and dopant densities for DX sites in AlGaAs:Sn

    NASA Astrophysics Data System (ADS)

    Lurio, L. B.; Hayes, T. M.; Pant, J.; Williamson, D. L.; Gibart, P.; Theis, T. N.

    1996-03-01

    X-ray absorption spectroscopy measurements using fluorescence detection have been performed on Sn dopant centers in Al_.33Ga_.77As:Sn. Absolute calibration of the fluorescence yield permits a comparison of dopant and charge carrier densities in the persistent photo-conducting (PPC) state. It is found that the ratio of dopants to carriers is significantly greater than 1:1. In order to understand this discrepancy, we have determined the average local environment of the dopants through analysis of the X-ray absorption fine structure. The reduced carrier density is explained by a combination of inactive donor sites and charge traps. Implications for the nature of the Sn donor sites will be discussed. This research is supported by NSF grant nos. DMR-9006956 and DMR-8902512. The measurements were made at SSRL which is funded by the DOE Office of Basic Energy Sciences and the NIH Biotechnology Research Resource Program.

  20. Radiation inactivation study of aminopeptidase: probing the active site

    NASA Astrophysics Data System (ADS)

    Jamadar, V. K.; Jamdar, S. N.; Mohan, Hari; Dandekar, S. P.; Harikumar, P.

    2004-04-01

    Ionizing radiation inactivated purified chicken intestinal aminopeptidase in media saturated with gases in the order N 2O>N 2>air. The D 37 values in the above conditions were 281, 210 and 198 Gy, respectively. OH radical scavengers such as t-butanol and isopropanol effectively nullified the radiation-induced damage in N 2O. The radicals (SCN) 2•-, Br 2•- and I 2•- inactivated the enzyme, pointing to the involvement of aromatic amino acids and cysteine in its catalytic activity. The enzyme exhibited fluorescence emission at 340 nm which is characteristic of tryptophan. The radiation-induced loss of activity was accompanied by a decrease in the fluorescence of the enzyme suggesting a predominant influence on tryptophan residues. The enzyme inhibition was associated with a marked increase in the Km and a decrease in the Vmax and kcat values, suggesting an irreversible alteration in the catalytic site. The above observations were confirmed by pulse radiolysis studies.

  1. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. Evaluation and use of sediment toxicity reference sites for statistical comparisons in regional assessments.

    PubMed

    Hunt, J W; Anderson, B S; Phillips, B M; Newman, J; Tjeerdema, R S; Fairey, R; Puckett, H M; Stephenson, M; Smith, R W; Wilson, C J; Taberski, K M

    2001-06-01

    Sediment reference sites were used to establish toxicity standards against which to compare results from sites investigated in San Francisco Bay (California, USA) monitoring programs. The reference sites were selected on the basis of low concentrations of anthropogenic chemicals, distance from active contaminant sources, location in representative hydrographic areas of the Bay, and physical features characteristic of depositional areas (e.g., fine grain size and medium total organic carbon [TOC]). Five field-replicated sites in San Francisco Bay were evaluated over three seasons. Samples from each site were tested with nine toxicity test protocols and were analyzed for sediment grain size and concentrations of trace metals, trace organics, ammonia, hydrogen sulfide, and TOC. The candidate sites were found to have relatively low concentrations of measured chemicals and generally exhibited low toxicity. Toxicity data from the reference sites were then used to calculate numerical tolerance limits to be used as threshold values to determine which test sites had significantly higher toxicity than reference sites. Tolerance limits are presented for four standard test protocols, including solid-phase sediment tests with the amphipods Ampelisca abdita and Eohaustorius estuarius and sea urchin Strongylocentrotus purpuratus embryo/larval development tests in pore water and at the sediment-water interface (SWI). Tolerance limits delineating the lowest 10th percentile (0.10 quantile) of the reference site data distribution were 71% of the control response for Ampelisca, 70% for Eohaustorius, 94% for sea urchin embryos in pore water, and 87% for sea urchins embryos exposed at the SWI. The tolerance limits are discussed in terms of the critical values governing their calculation and the management implications arising from their use in determining elevated toxicity relative to reference conditions.

  5. An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

    SciTech Connect

    Guidinger, P.F.; Nowak, T. )

    1991-09-10

    The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 {plus minus} 0.025 M{sup {minus}1} min{sup {minus}1}. Inactivation by pyridoxal 5{prime}-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7,700 {plus minus} 860 m{sup {minus}1} min{sup {minus}1}. Treatment of the enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of k{sub obs} vs pH suggests this active-site lysine has a pK{sub a} of 8.1 and a pH-independent rate constant of inactivation of 47,700 m{sup {minus}1} min{sup {minus}1}. Proton relaxation rate measurements suggest that pyridoxal 5{prime}-phosphate modification alters binding of the phosphate-containing substrates. {sup 31}P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme {center dot}Mn{sup 2+}{center dot}substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5{prime}-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.

  6. A comparison of small, affordable seismic sources at the Ketzin CO2 storage site, Germany

    NASA Astrophysics Data System (ADS)

    Sopher, Daniel; Juhlin, Christopher; Huang, Fei; Ivandic, Monika; Lueth, Stefan

    2014-05-01

    Seismic methods have proven to be effective for monitoring the movement and location of injected CO2 within deep saline aquifers. However, a disadvantage of seismic monitoring is the high costs associated with many repeat seismic surveys as part of a long term monitoring strategy of a CO2 storage site. As the cost for the use of the seismic source is often a significant part of the overall survey cost, affordable, smaller sources would increase the potential feasibility of a long term seismic monitoring strategy. A comparison of three land seismic sources is performed at the Ketzin CO2 storage site, Germany. Two of these sources (Vibsist 500 and Bobcat drop hammer) can be considered to be smaller and more affordable sources than those conventionally used in the seismic monitor surveys at Ketzin. In this study these smaller sources are compared to a larger more conventional Vibsist 3000 source. The subsurface target for the three sources in this comparison is the CO2 storage reservoir for the Ketzin site, located within the Triassic Stuttgart formation, which lies at a depth of approximately 600m/500ms. Two of the sources are Swept Impact (SIST) type courses (Vibsist 500 and 3000) which use hydraulic concrete breaking hammers. The third source uses a concrete breaking drop hammer tool mounted on a Bobcat loader. Data were collected along a 984m long profile with 24m receiver spacing and 12m shot spacing in 2011, 2012 and 2013 using the three different sources. A quantitative and qualitative comparison of the raw data from the three sources was performed in order to assess their relative performance. Frequency content, signal to noise ratio and penetration depth curves were calculated for the raw data. Data from the three sources was also processed using a conventional workflow to produce stacked sections which were compared. Based on the results from this study the Bobcat drop hammer source appears to perform better than the Vibsist 500 source. However both of the

  7. Direct site-specific glycoform identification and quantitative comparison of glycoprotein therapeutics: imiglucerase and velaglucerase alfa.

    PubMed

    Ye, Hongping; Hill, John; Gucinski, Ashley C; Boyne, Michael T; Buhse, Lucinda F

    2015-03-01

    Gaucher disease, the most common lysosomal metabolic disorder, can be treated with enzyme replacement therapy (ERT). Recombinant human glucocerebrosidase imiglucerase (Cerezyme(®)), produced in Chinese hamster ovary cells, has been used for ERT of Gaucher disease for 20 years. Another recombinant glucocerebrosidase velaglucerase alfa (VPRIV), expressed in a human fibroblast cell line, was approved by the US Food and Drug Administration in 2010. The amino acid sequence difference at residue 495 of these two products is well documented. The overall N-linked qualitative glycan composition of these two products has also been reported previously. Herein, employing our recently developed approach utilizing isobaric tandem mass tag (TMT) labeling and an LTQ Orbitrap XL electron transfer dissociation (ETD) hybrid mass spectrometer, the site-specific glycoforms of these products were identified with ETD and collision-induced dissociation (CID) spectra. The quantitative comparison of site-specific glycans was achieved utilizing higher-energy collisional dissociation (HCD) spectra with a NanoMate used as both a fraction collector and a sample introduction device. From the trypsin-digested mixture of these two products, over 90 glycopeptides were identified by accurate mass matching. In addition to those previously reported, additional glycopeptides were detected with moderate abundance. The relative amount of each glycoform at a specific glycosylation site was determined based on reporter signal intensities of the TMT labeling reagents. This is the first report of site-specific simultaneous qualitative and quantitative comparison of glycoforms for Cerezyme(®) and VPRIV. The results demonstrate that this method could be utilized for biosimilarity determination and counterfeit identification of glycoproteins. PMID:25501675

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. Comparison of Activity Determination of Radium 226 in FUSRAP Soil using Various Energy Lines - 12299

    SciTech Connect

    Tucker, Brian; Donakowski, Jough; Hays, David

    2012-07-01

    Gamma spectroscopy is used at the Formerly Utilized Sites Remedial Action Program (FUSRAP) Maywood Superfund Site as the primary radioanalytical tool for quantization of activities of the radionuclides of concern in site soil. When selecting energy lines in gamma spectroscopy, a number of factors are considered including assumptions concerning secondary equilibrium, interferences, and the strength of the lines. The case of the Maywood radionuclide of concern radium-226 (Ra-226) is considered in this paper. At the FUSRAP Maywood Superfund Site, one of the daughters produced from radioactive decay of Ra-226, lead-214 (Pb- 214), is used to quantitate Ra-226. Another Ra-226 daughter, bismuth-214 (Bi-214), also may be used to quantitate Ra-226. In this paper, a comparison of Ra-226 to Pb-214 activities and Ra-226 to Bi-214 activities, obtained using gamma spectrometry for a large number of soil samples, was performed. The Pb-214, Bi-214, and Ra-226 activities were quantitated using the 352 kilo electron volt (keV), 609 keV, and 186 keV lines, respectively. The comparisons were made after correcting the Ra-226 activities by a factor of 0.571 and both ignoring and accounting for the contribution of a U-235 interfering line to the Ra-226 line. For the Pb-214 and Bi-214 activities, a mean in-growth factor was employed. The gamma spectrometer was calibrated for efficiency and energy using a mixed gamma standard and an energy range of 59 keV to 1830 keV. The authors expect other sites with Ra-226 contamination in soil may benefit from the discussions and points in this paper. Proper use of correction factors and comparison of the data from three different gamma-emitting radionuclides revealed agreement with expectations and provided confidence that using such correction factors generates quality data. The results indicate that if contamination is low level and due to NORM, the Ra-226 can be measured directly if corrected to subtract the contribution from U-235. If there is

  15. Development and comparison of five site-specific biosphere models for safety assessment of radioactive waste disposal.

    PubMed

    Pröhl, G; Olyslaegers, G; Kanyar, B; Pinedo, P; Bergström, U; Mobbs, S; Eged, K; Katona, T; Simón, I; Hallberg, U B; Chen, Q; Kowe, R; Zeevaert, T

    2005-12-01

    This paper describes the development and application of site-specific biosphere models that might be used for assessment of potential exposures in the framework of performance assessment studies of nuclear waste disposals. Model development follows the Reference Biosphere Methodology that has been set up in the framework of the BIOMASS study. In this paper, the application is to real sites at five European locations for which environmental and agricultural conditions have been described and characterised. For each of the sites a biosphere model has been developed specifically assuming a release of radionuclides to waters that are used by humans, for example as drinking water for humans and cattle and as irrigation water. Among the ingestion pathways, the intakes of drinking water, cereals, leafy vegetables, potatoes, milk, beef and freshwater fish are included in all models. Annual individual doses were calculated, and uncertainties in the results were estimated by means of stochastic calculations. To enable a comparison, all results were normalised to an activity concentration in groundwater of 1 Bq m(-3) for each of the radionuclides considered ((36)Cl, (79)Se, (99)Tc, (129)I, (135)Cs, (226)Ra, (231)Pa, (230)Th, (237)Np, (239)Pu, and (238)U), i.e. those that are usually most relevant in performance assessment studies of nuclear waste disposals. Although the results do not give answers in absolute terms on potential future exposures, they indicate the spectrum of exposures that might occur in different environments and specify the interaction of environmental conditions, human habits and potential exposure.

  16. 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

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

    PubMed

    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.

  18. 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

  19. 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

  20. 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.

  1. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    PubMed

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe

    2016-01-01

    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity. PMID:27443004

  2. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    PubMed

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe

    2016-01-01

    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity.

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

    PubMed

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

    2015-02-27

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

  4. Metals in the active site of native protein phosphatase-1.

    PubMed

    Heroes, Ewald; Rip, Jens; Beullens, Monique; Van Meervelt, Luc; De Gendt, Stefan; Bollen, Mathieu

    2015-08-01

    Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone. PMID:25890482

  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. Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease*

    PubMed Central

    da Palma, Joel Ramos; Burri, Dominique Julien; Oppliger, Joël; Salamina, Marco; Cendron, Laura; de Laureto, Patrizia Polverino; Seidah, Nabil Georges; Kunz, Stefan; Pasquato, Antonella

    2014-01-01

    The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B′/B followed by the herein newly identified C′/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B′/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1–P8) and P1′ are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates. PMID:25378398

  7. Seasonal factors affecting surfactant biodegradation in Antarctic coastal waters: comparison of a polluted and pristine site.

    PubMed

    George, Alison L

    2002-05-01

    This report is the first seasonal study of anthropogenic pollutant biodegradation rates in Antarctic coastal waters. The capacity of surface waters from Rothera Research Station, Adelaide Island, Antarctica, to biodegrade the anionic surfactant sodium dodecyl sulphate (SDS) was quantified in biodegradation tests from April 1988 to January 1999. Large temporal differences in the persistence of SDS were observed. In mid-winter (July), the SDS-biodegradation half life was twice that measured in mid-summer (January), despite small temperature differences (up to 2.45 degrees C). Comparisons between water from a pristine site and a site receiving grey-waste water from the station showed that some acclimation to SDS was occurring in the contaminated water. This resulted in SDS half lives up to to approximately 80 h shorter in the polluted water compared with the pristine site in the summer months when a large population of SDS-degrading bacteria had developed. Biodegradation half lives in Antarctic coastal waters (160-460 h) were generally far higher than those observed in temperate waters.

  8. The effects of an engineered cation site on the structure, activity, and EPR properties of cytochrome c peroxidase.

    PubMed

    Bonagura, C A; Sundaramoorthy, M; Bhaskar, B; Poulos, T L

    1999-04-27

    Earlier work [Bonagura et al. (1996) Biochemistry 35, 6107] showed that the K+ site found in the proximal pocket of ascorbate peroxidase (APX) could be engineered into cytochrome c peroxidase (CCP). Binding of K+ at the engineered site results in a loss in activity and destabilization of the CCP compound I Trp191 cationic radical owing to long-range electrostatic effects. The engineered CCP mutant crystal structure has been refined to 1.5 A using data obtained at cryogenic temperatures which provides a more detailed basis for comparison with the naturally occurring K+ site in APX. The characteristic EPR signal associated with the Trp191 radical becomes progressively weaker as K+ is added, which correlates well with the loss in enzyme activity as [K+] is increased. These results coupled with stopped-flow studies support our earlier conclusions that the loss in activity and EPR signal is due to destabilization of the Trp191 cationic radical. PMID:10220341

  9. Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

    SciTech Connect

    Nicholas, R.A.; Suzuki, H.; Hirota, Y.; Strominger, J.L.

    1985-07-02

    This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with (/sup 14/C)penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the (/sup 14/C)penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.

  10. Characterization and sequencing of an active-site cysteine-containing peptide from the xylanase of a thermotolerant Streptomyces.

    PubMed

    Keskar, S S; Rao, M B; Deshpande, V V

    1992-02-01

    The kinetics of chemical modification of the xylanase from a thermotolerant Streptomyces T7 indicated the involvement of 1 mol of cysteine residue/mol of enzyme [Keskar, Srinivasan & Deshpande (1989) Biochem. J. 261, 49-55]. The chromophoric reagent N-(2,4-dinitroanilino)maleimide (DAM) reacts covalently with thiol groups of xylanase with complete inactivation. Protection against inactivation was provided by the substrate (xylan). The purified xylanase that had been modified with DAM was digested with pepsin and the peptides were purified by gel filtration followed by peptide mapping. The active-site peptide was distinguished from the other thiol-containing peptides by comparison of the peptides generated by labelling the enzyme in the presence and in the absence of the substrate. The peptide mapping of the modified enzyme in the absence of xylan showed three yellow peptides, whereas in the presence of xylan only two yellow peptides were detected. The active-site peptide protected by the substrate failed to form the complex with DAM. The modified active-site peptide was isolated and sequenced. Gas-phase sequencing provided the following sequence: Ser-Val-Ile-Met-Xaa-Ile-Asp-His-Ile-Arg-Phe. This is the first report on the isolation and sequencing of the active-site peptide from a xylanase. The comparison of reactive cysteine-containing peptide sequence with the catalytic regions of other glucanases revealed the presence of a conserved aspartic acid residue.

  11. 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

  12. Comparison of alternative MS/MS and bioinformatics approaches for confident phosphorylation site localization.

    PubMed

    Wiese, Heike; Kuhlmann, Katja; Wiese, Sebastian; Stoepel, Nadine S; Pawlas, Magdalena; Meyer, Helmut E; Stephan, Christian; Eisenacher, Martin; Drepper, Friedel; Warscheid, Bettina

    2014-02-01

    Over the past years, phosphoproteomics has advanced to a prime tool in signaling research. Since then, an enormous amount of information about in vivo protein phosphorylation events has been collected providing a treasure trove for gaining a better understanding of the molecular processes involved in cell signaling. Yet, we still face the problem of how to achieve correct modification site localization. Here we use alternative fragmentation and different bioinformatics approaches for the identification and confident localization of phosphorylation sites. Phosphopeptide-enriched fractions were analyzed by multistage activation, collision-induced dissociation and electron transfer dissociation (ETD), yielding complementary phosphopeptide identifications. We further found that MASCOT, OMSSA and Andromeda each identified a distinct set of phosphopeptides allowing the number of site assignments to be increased. The postsearch engine SLoMo provided confident phosphorylation site localization, whereas different versions of PTM-Score integrated in MaxQuant differed in performance. Based on high-resolution ETD and higher collisional dissociation (HCD) data sets from a large synthetic peptide and phosphopeptide reference library reported by Marx et al. [Nat. Biotechnol. 2013, 31 (6), 557-564], we show that an Andromeda/PTM-Score probability of 1 is required to provide an false localization rate (FLR) of 1% for HCD data, while 0.55 is sufficient for high-resolution ETD spectra. Additional analyses of HCD data demonstrated that for phosphotyrosine peptides and phosphopeptides containing two potential phosphorylation sites, PTM-Score probability cutoff values of <1 can be applied to ensure an FLR of 1%. Proper adjustment of localization probability cutoffs allowed us to significantly increase the number of confident sites with an FLR of <1%.Our findings underscore the need for the systematic assessment of FLRs for different score values to report confident modification site

  13. 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

  14. Comparison of Aerosol Properties Within and Above the ABL at the ARM Program's SGP Site

    SciTech Connect

    Delle Monache, L

    2002-05-01

    The goal of this thesis is to determine under what conditions, if any, measurements of aerosol properties made at the Earth's surface are representative of aerosol properties within the column of air above the surface. This thesis will use data from the Atmospheric Radiation Measurement (ARM) site at the Southern Great Plains (SGP) which is the only location in the world where ground-based and in situ airborne measurements are made on a routine basis. All flight legs in the one-year period from March 2000-March 2001 were categorized as either within or above the atmospheric boundary layer using an objective mixing height determination technique. The correlations between the aerosol properties measured at the surface and the measured within and above the ABL were then computed. The conclusion of this comparison is that the aerosol extensive and intensive properties measured at the surface are representative of values within the ABL, but not within the free atmosphere.

  15. New Particle Formation in the Remote Troposphere: A Comparison of Observations at Various Sites

    NASA Astrophysics Data System (ADS)

    Weber, R. J.; McMurry, P. H.; Mauldin, R. L., III; Tanner, D. J.; Eisele, F. L.; Clarke, A. D.; Kapustin, V. N.

    Measurements show that new particles are formed by homogenous nucleation over a wide range of conditions in the remote troposphere. In our studies, large nucleation events are found exclusively in regions of enhanced sulfuric acid vapor (H2SO4g) concentrations, with maximum concentrations never exceeding 5×107 molecules cm-3. Although these data suggest that H2SO4g participated, comparisons between ambient conditions in regions of nucleation to conditions necessary for binary H2SO4 water (H2O) nucleation indicate that the mechanism may vary with elevation. In remote marine regions, at altitudes greater than ˜4 km above sea level, observations of nucleation in clear air along cloud perimeters are in fair agreement with current classical binary nucleation models. In these regions, the low temperatures associated with high altitudes may produce sufficiently saturated H2SO4 for the production of new H2SO4/H2O particles. However, uncertainties with current binary nucleation models limit decisive comparisons. In warmer regions, closer to the earth's surface, measured H2SO4 concentrations are clearly insufficient for binary nucleation. Conditions at these sites are similar to those observed in an earlier study where there was circumstantial evidence for a ternary mechanism involving H2SO4, H2O, and ammonia (NH3) [Weber et al., 1998], suggesting that this may be a significant route for particle production at lower altitudes where surface-derived species, like NH3, are more apt to participate.

  16. Detecting Local Ligand-Binding Site Similarity in Non-Homologous Proteins by Surface Patch Comparison

    PubMed Central

    Sael, Lee; Kihara, Daisuke

    2012-01-01

    Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. PMID:22275074

  17. Prioritizing conservation activities using reserve site selection methods and population viability analysis.

    PubMed

    Newbold, Stephen C; Siikamäki, Juha

    2009-10-01

    In recent years a large literature on reserve site selection (RSS) has developed at the interface between ecology, operations research, and environmental economics. Reserve site selection models use numerical optimization techniques to select sites for a network of nature reserves for protecting biodiversity. In this paper, we develop a population viability analysis (PVA) model for salmon and incorporate it into an RSS framework for prioritizing conservation activities in upstream watersheds. We use spawner return data for three closely related salmon stocks in the upper Columbia River basin and estimates of the economic costs of watershed protection from NOAA to illustrate the framework. We compare the relative cost-effectiveness of five alternative watershed prioritization methods, based on various combinations of biological and economic information. Prioritization based on biological benefit-economic cost comparisons and accounting for spatial interdependencies among watersheds substantially outperforms other more heuristic methods. When using this best-performing prioritization method, spending 10% of the cost of protecting all upstream watersheds yields 79% of the biological benefits (increase in stock persistence) from protecting all watersheds, compared to between 20% and 64% for the alternative methods. We also find that prioritization based on either costs or benefits alone can lead to severe reductions in cost-effectiveness. PMID:19831069

  18. Comparison of Aerosol Properties within and above the ABL at the ARM Program's SGP Site

    SciTech Connect

    Monache, L.D.; Perry, K.D.; Cederwall, R.T.

    2002-02-26

    The goal of this study was to determine under what conditions, if any, measurements of aerosol properties made at the Earth's surface are representative of the aerosol properties within the column of air above the surface. This project used data from the Atmospheric Radiation Measurement (ARM) site at the Southern Great Plains (SGP) site (Stokes and Schwartz 1994), which is one of the only locations in the world where ground-based and in situ airborne measurements of atmospheric aerosol are made on a routine basis. All flight legs in the one-year period from March 2000 to March 2001 were categorized as either within or above the atmospheric boundary layer (ABL) using an objective mixing height determination technique. The correlations between the aerosol properties measured at the surface and those measured within and above the ABL were then computed. The conclusion of this comparison is that the aerosol extensive properties (those that depend upon the amount of aerosol that is present in the atmosphere, i.e., either the number or mass concentrations), and intensive properties (those that do not depend upon the amount of aerosol present) measured at the surface are representative of values within the ABL, but not within the free atmosphere.

  19. A COMPARISON OF HANFORD AND SAVANNAH RIVER SITE HIGH-LEVEL WASTES

    SciTech Connect

    HILL RC PHILIP; REYNOLDS JG; RUTLAND PL

    2011-02-23

    This study is a simple comparison of high-level waste from plutonium production stored in tanks at the Hanford and Savannah River sites. Savannah River principally used the PUREX process for plutonium separation. Hanford used the PUREX, Bismuth Phosphate, and REDOX processes, and reprocessed many wastes for recovery of uranium and fission products. Thus, Hanford has 55 distinct waste types, only 17 of which could be at Savannah River. While Hanford and Savannah River wastes both have high concentrations of sodium nitrate, caustic, iron, and aluminum, Hanford wastes have higher concentrations of several key constituents. The factors by which average concentrations are higher in Hanford salt waste than in Savannah River waste are 67 for {sup 241}Am, 4 for aluminum, 18 for chromium, 10 for fluoride, 8 for phosphate, 6 for potassium, and 2 for sulfate. The factors by which average concentrations are higher in Hanford sludges than in Savannah River sludges are 3 for chromium, 19 for fluoride, 67 for phosphate, and 6 for zirconium. Waste composition differences must be considered before a waste processing method is selected: A method may be applicable to one site but not to the other.

  20. Comparison of adICPR vs. UNET to evaluate surface water issues at a CERCLA site

    SciTech Connect

    Cockcroft, B.F.; Campbell, C.M.

    1994-12-31

    A model of the surface water flow regime for the site was developed to evaluate the recommended remedial action alternative. The widely used and accepted Corps of Engineers` HEC-1 and HEC-2 computer programs were used for the traditional hydrologic and steady-state hydraulic analyses. Evaluation of the dynamic (i.e., unsteady) flows through the interconnected low areas was not as straight-forward and consensus on which program to use was an issue. Thus, two computer programs were used; the privately developed Advanced Interconnected Channel and Pond Routing (adICPR) program and UNET -- One-Dimensional Unsteady Flow Through a Full Network of Open Channels developed by the US Army Corps of Engineers` Hydrologic Engineering Center. The two unsteady flow programs were interchanged in the overall surface water model of the site to evaluate the recommended remedial action alternatives. This paper presents a comparison of the two unsteady flow programs and the corresponding results. Most noteworthy, however, this paper identifies and demonstrates a lesson for future evaluations to improve the cost effectiveness of the CERCLA process. The lesson being that the selection of, and the consensus on, design criteria and objectives play a more critical and influential role in the overall evaluation and selection of the remedial action alternative than the differences between the two computer programs.

  1. Comparison of the effects of pollution on the marine bivalve Donax trunculus in the vicinity of polluted sites with specimens from a clean reference site (Mediterranean Sea).

    PubMed

    Yawetz, Aminadav; Fishelson, Lev; Bresler, Vladimir; Manelis, Rami

    2010-02-01

    The physiological and biochemical characteristics of tissue samples of the marine mussel, Donax trunculus, from an oil-polluted site (Qiryat Yam) and from a site adjacent to an industrial factory (Frutarom) producing polyvinyl chloride (PVC) were compared with samples from a clean reference site (Akko). All sites were located along the sandy shores of the Israeli Mediterranean Sea. The mussels from the oil-polluted site showed increased activity of the system of active transport of organic anions (SATOA) in the gills and the renocardial organ and also of the multixenobiotic resistance transporter (MXR) in the gills. In contrast, samples collected near the PVC factory showed a decrease in SATOA activity and no increase in the activity of MXR in the gills. Specimens from the reference site demonstrated a redox state of equilibrium between energy production and utilization, while in Donax from both the oil polluted and the PVC-polluted sites, the mitochondrial redox state reflected intensive consumption of energy. No significant changes were found in the activity of reduced glutathione s-transferase (GST) in the cytosolic fraction of the digestive gland of Donax collected from any of the three sites. The data demonstrate a differential increase in the anti-chemical defense systems and an intensification of energy metabolism in the mussels exposed to pollution.

  2. Structural difference at the active site of dibucaine resistant variant of human plasma cholinesterase.

    PubMed Central

    Muensch, H; Yoshida, A; Altland, K; Jensen, W; Goedde, H W

    1978-01-01

    Human plasma cholinesterase from five different genotypes -- E1U E1U, E1U E1A, E1A E1A, E1U E1S, E1A E1S, and E1U E1U C5+ -- was purified 8,000 fold from serum by a two-step procedure involving chromatography on DEAE-cellulose and preparative disc electrophoresis. The esterases were labeled with diisopropyl-1, 3-C14-fluorophosphate (DFP) aminoethylated, and digested by trypsin. The trytic digests were subjected to high voltage electrophoresis, and the radioactive peptides were detected by radioautography. Comparison of the peptides revealed different electrophoretic mobilities of the usual and atypical (dibucaine resistant) plasma cholinesterase peptides. The results are consistent with a structural abnormality of the active center in the variant enzyme. No difference was observed an the esteratic site of the enzyme with C5 component. Images Fig. 1 PMID:677127

  3. 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

  4. 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.

  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. Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

    PubMed

    Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

    2016-09-01

    A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

  7. 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

  8. Non-invasive shallow seismic source comparison for hazardous waste site investigations

    SciTech Connect

    Doll, W.E.; Miller, R.D.; Xia, J.

    1994-12-31

    Many commonly used shallow seismic sources are unacceptable for hazardous waste site investigations because they risk exhumation of contaminants in the soil, they add contaminants (e.g. lead) which are not allowed by regulations, or they add new migration paths for contaminants. Furthermore, recently developed high frequency vibrators for shallow investigations could be more effective at some sites than non-invasive impulsive sources because of their ability to tailor the source spectrum and reduce interference. The authors show preliminary results of a comparison test of eight non-invasive impulsive and swept sources in preparation for seismic reflection profiling on the Oak Ridge Reservation, Tennessee. Well log data are used to determine geologic contacts and to generate synthetic seismograms for the site. Common midpoint (CMP) seismic data for each source were collected at 95 geophone groups from 125 shot points along a 400m test line. Hydrophone data were obtained at 1.5m spacing between 61m and 133m depth in a hole near the center of the CMP line. As of March, 1994, brute stacks have been completed for three of the eight sources. Depth penetration is demonstrated in brute stacks and shot gathers, which show a 200ms reflector for all of the sources tested along portions of the line. Source effectiveness will also be evaluated by comparing images of several shallower reflectors (40--150ms) which are apparent in many of the records. Imaging of these reflectors appears to depend upon the ability of the source to generate sufficient high frequency energy (>100 Hz).

  9. Tomogram-based comparison of geostatistical models: Application to the Macrodispersion Experiment (MADE) site

    NASA Astrophysics Data System (ADS)

    Linde, Niklas; Lochbühler, Tobias; Dogan, Mine; Van Dam, Remke L.

    2015-12-01

    We propose a new framework to compare alternative geostatistical descriptions of a given site. Multiple realizations of each of the considered geostatistical models and their corresponding tomograms (based on inversion of noise-contaminated simulated data) are used as a multivariate training image. The training image is scanned with a direct sampling algorithm to obtain conditional realizations of hydraulic conductivity that are not only in agreement with the geostatistical model, but also honor the spatially varying resolution of the site-specific tomogram. Model comparison is based on the quality of the simulated geophysical data from the ensemble of conditional realizations. The tomogram in this study is obtained by inversion of cross-hole ground-penetrating radar (GPR) first-arrival travel time data acquired at the MAcro-Dispersion Experiment (MADE) site in Mississippi (USA). Various heterogeneity descriptions ranging from multi-Gaussian fields to fields with complex multiple-point statistics inferred from outcrops are considered. Under the assumption that the relationship between porosity and hydraulic conductivity inferred from local measurements is valid, we find that conditioned multi-Gaussian realizations and derivatives thereof can explain the crosshole geophysical data. A training image based on an aquifer analog from Germany was found to be in better agreement with the geophysical data than the one based on the local outcrop, which appears to under-represent high hydraulic conductivity zones. These findings are only based on the information content in a single resolution-limited tomogram and extending the analysis to tracer or higher resolution surface GPR data might lead to different conclusions (e.g., that discrete facies boundaries are necessary). Our framework makes it possible to identify inadequate geostatistical models and petrophysical relationships, effectively narrowing the space of possible heterogeneity representations.

  10. Comparison of Methods to Obtain Force-Field Parameters for Metal Sites.

    PubMed

    Hu, LiHong; Ryde, Ulf

    2011-08-01

    We have critically examined and compared various ways to obtain standard harmonic molecular mechanics (MM) force-field parameters for metal sites in proteins, using the 12 most common Zn(2+) sites as test cases. We show that the parametrization of metal sites is hard to treat with automatic methods. The choice of method is a compromise between speed and accuracy and therefore depends on the intended use of the parameters. If the metal site is not of central interest in the investigation, for example, a structural metal far from the active site, a simple and fast parametrization is normally enough, using either a nonbonded model with restraints or a bonded parametrization based on the method of Seminario. On the other hand, if the metal site is of central interest in the investigation, a more accurate method is needed to give quantitative results, for example, the method by Norrby and Liljefors. The former methods are semiautomatic and can be performed in seconds, once a quantum mechanical (QM) geometry optimization and frequency calculation has been performed, whereas the latter method typically takes several days and requires significant human intervention. All approaches require a careful selection of the atom types used. For a nonbonded model, standard atom types can be used, whereas for a bonded model, it is normally wise to use special atom types for each metal ligand. For accurate results, new atom types for all atoms in the metal site can be used. Atomic charges should also be considered. Typically, QM restrained electrostatic potential charges are accurate and easy to obtain once the QM calculation is performed, and they allow for charge transfer within the complex. For negatively charged complexes, it should be checked that hydrogen atoms of the ligands get proper charges. Finally, water ligands pose severe problems for bonded models in force fields that ignore nonbonded interactions for atoms separated by two bonds. Complexes with a single water ligand

  11. Associations between Restriction Site Polymorphism and Enzyme Activity Variation for Esterase 6 in Drosophila Melanogaster

    PubMed Central

    Game, A. Y.; Oakeshott, J. G.

    1990-01-01

    Thirty-five nucleotide polymorphisms were found in a 21.5-kbp region including the Est6 locus among 42 isoallelic lines extracted from a single natural population of Drosophila melanogaster. The heterozygosity per nucleotide pair was estimated to be 0.010 overall, but was lower in sequences hybridizing to transcripts than in those not hybridizing to transcripts. Eleven of 36 pairwise comparisons among the nine most common polymorphisms showed significant gametic disequilibrium. Four of these polymorphisms were also significantly associated with the major EST6-F/EST6-S allozyme polymorphism. Significant disequilibrium was generally restricted to polymorphisms less than 1-2 kbp apart. Previously reported measures of EST6 activity in virgin adult females proved not to be significantly associated with any of the six most common nucleotide polymorphisms located in the Est6 coding region or the 1.5 kbp immediately 5'. However, the 11 haplotypes for five of these polymorphisms that lie in the 1.5-kbp 5' region could explain about half of the previously reported variation among the lines for both EST6 activity and the amount of EST6 protein in virgin adult males. One particular polymorphism, for a RsaI site 530 bp 5' of the initiation codon, could explain 21% of the male activity variation among lines. This site is embedded in a large palindrome and we suggest that sequences including or close to this site may be involved in the regulation of EST6 synthesis in the ejaculatory duct of the adult male. PMID:1981760

  12. Comparison of 1-Site and 2-Site Phacotrabeculectomy in the Small Adult Eyes With Concomitant Cataract and Glaucoma

    PubMed Central

    Xia, Xiaobo; Tian, Ying; Wu, Zhenkai; Wen, Dan; Song, Weitao

    2016-01-01

    Abstract The aim of this study was to compare the outcomes after phacotrabeculectomy at 1 or 2 sites in the small adult eyes with concomitant cataract and glaucoma. Patients who had 1-site (n = 26) or 2-site (n = 14) phacotrabeculectomy over a 4-year period at an eye surgery center were included. Eighteen eyes of 18 patients with glaucoma using any 1 prostaglandin analogue (latanoprost, travoprost, or bimatoprost) were compared with 8 normal control patients. The records of patients were reviewed, and intraocular pressure, best-corrected visual acuity, axial length, anterior chamber depth, corneal endothelial cell (CEC) density, Diopter were measured. The outcome was compared with postoperative and preoperative measurements for 3-month follow-ups. The follow-up time was 3 months. There was no difference between the operations in improving best-corrected visual acuity, lowering intraocular pressure, shortening axial length, and deepening anterior chamber depth. However, 2-site surgery was associated with significantly more CEC loss and refractive error. Postoperative complications were not different between the 2 groups. The CEC loss and the refractive error in 2-site group were higher than that of 1-site group. One-site surgery seems to cause less CEC damage and refractive error than the 2-site operation during the follow-up time of 3 months. PMID:26844453

  13. 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...

  14. 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...

  15. 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...

  16. 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...

  17. 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...

  18. 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.

  19. Control of active sites in selective flocculation: III -- Mechanism of site blocking

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    It has been shown in Parts I and II of this paper that heteroflocculation can be controlled by poisoning the sites for flocculant adsorption using a site blocking agent (SBA). An efficient SBA was determined to be the lower molecular weight fraction of the flocculant. In this paper, the underlying mechanism of SBA action is described. Also, the mathematical model detailed in Part I is used to determine the effect of different SBAs on apatite-dolomite separation efficiency. It has been demonstrated that the depression in flocculation is directly related to the site blocking parameter ([bar [Phi

  20. Dynamically achieved active site precision in enzyme catalysis.

    PubMed

    Klinman, Judith P

    2015-02-17

    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.

  1. Comparison of donor-site morbidity and satisfaction between anterolateral thigh and parascapular free flaps in the same patient.

    PubMed

    Fischer, Sebastian; Klinkenberg, Marek; Behr, Bjoern; Hirsch, Tobias; Kremer, Thomas; Hernekamp, Frederick; Kolbenschlag, Jonas; Lehnhardt, Marcus; Kneser, Ulrich; Daigeler, Adrien

    2013-10-01

    The purpose of this study was to compare donor-site morbidity after anterolateral thigh (ALT) and parascapular (PS) free flap harvest in the same patient. A total of 13 patients were included in this study. Because of initial flap loss, each patient received ALT as well as PS free flap transplantation. A total of 10 patients were available for follow-up. The average follow-up time was 44.5 months. Besides physical examination, range of motion and scar dimensions were assessed to objectify donor-site deficiencies. The subjective donor-site morbidity was assessed by the patients using a self-report questionnaire. In addition, patients were requested to state their donor-site preference. ALT donor site revealed more sensitivity deficiencies compared with the PS harvest site (8 vs. 4). The latter provoked less functional impairments (1 vs. 2). Scar dimensions were larger at PS harvest site (25.8 × 4.3 cm vs. 23.3 × 3.6 cm). Patients' satisfaction was in favor of the PS donor site (1.9 vs. 2.7). Among the 10 patients, 7 patients preferred the PS and 3 patients preferred the ALT donor site. Comparison of donor-site morbidities in the same patient reveals a valuable tool to diminish individual bias. Despite the low number of cases, we were able to demonstrate the superiority of PS compared with ALT donor sites.The Level of Evidence of the study is III.

  2. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  3. Monoclonal antibody against the active site of caeruloplasmin and the ELISA system detecting active caeruloplasmin.

    PubMed

    Hiyamuta, S; Ito, K

    1994-04-01

    Serum caeruloplasmin deficiency is a characteristic biochemical abnormality found in patients with Wilson's disease, but the mechanism of this disease is unknown. Although the phenylenediamine oxidase activity of serum caeruloplasmin is markedly low in patients with Wilson's disease, mRNA of caeruloplasmin exists to some extent. To investigate the deficiency of caeruloplasmin oxidase activity in Wilson's disease, we generated 14 monoclonal antibodies (MAbs) and selected ID1, which had the strongest reactivity, and ID2, which had neutralizing ability. We also established a system to measure active caeruloplasmin specifically using these MAbs. These MAbs and the system will be useful tools in analyzing the active site of caeruloplasmin in patients with Wilson's disease.

  4. 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.

  5. Control of active sites in selective flocculation: II -- Role of site blocking agents

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Control of heteroflocculation using a lower molecular weight fraction of the flocculant as a site blocking agent is demonstrated in the apatite-dolomite-polyethylene oxide system. The most effective SBA (site blocking agent) was determined to be the highest molecular weight fraction of the flocculant itself which was not capable of flocculating any of the components of the mixture. In the presence of the SBA, flocculant adsorption decreased significantly on apatite particles, thereby inhibiting coflocculation.

  6. The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase.

    PubMed

    Anufrieva, Natalya V; Faleev, Nicolai G; Morozova, Elena A; Bazhulina, Natalia P; Revtovich, Svetlana V; Timofeev, Vladimir P; Tkachev, Yaroslav V; Nikulin, Alexei D; Demidkina, Tatyana V

    2015-09-01

    In the spatial structure of methionine γ-lyase (MGL, EC 4.4.1.11) from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate "handle" of pyridoxal 5'-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96Å resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and β-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-β- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and β-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

  7. COMPARISON OF THE SPECIATED VOC COMPOSITION AT SELECTED HOUSTON, TEXAS AREA SITES FOR THE YEARS 1978 AND 2000

    EPA Science Inventory

    Comparisons are presented for the C2-C12 volatile organic compounds (VOCs) observed at Houston area sites used for ambient air monitoring during special ozone field study programs conducted in 1978 and 2000. In 1978 the special study called the Houston Ozone Modeling Study cons...

  8. Comparison of template registration methods for multi-site meta-analysis of brain morphometry

    NASA Astrophysics Data System (ADS)

    Faskowitz, Joshua; de Zubicaray, Greig I.; McMahon, Katie L.; Wright, Margaret J.; Thompson, Paul M.; Jahanshad, Neda

    2016-03-01

    Neuroimaging consortia such as ENIGMA can significantly improve power to discover factors that affect the human brain by pooling statistical inferences across cohorts to draw generalized conclusions from populations around the world. Voxelwise analyses such as tensor-based morphometry also allow an unbiased search for effects throughout the brain. Even so, such consortium-based analyses are limited by a lack of high-powered methods to harmonize voxelwise information across study populations and scanners. While the simplest approach may be to map all images to a single standard space, the benefits of cohort-specific templates have long been established. Here we studied methods to pool voxel-wise data across sites using templates customized for each cohort but providing a meaningful common space across all studies for voxelwise comparisons. As non-linear 3D MRI registrations represent mappings between images at millimeter resolution, we need to consider the reliability of these mappings. To evaluate these mappings, we calculated test-retest statistics on the volumetric maps of expansion and contraction. Further, we created study-specific brain templates for ten T1-weighted MRI datasets, and a common space from four study-specific templates. We evaluated the efficacy of using a two-step registration framework versus a single standard space. We found that the two-step framework more reliably mapped subjects to a common space.

  9. Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders.

    PubMed

    Gómez-Marín, Carlos; Tena, Juan J; Acemel, Rafael D; López-Mayorga, Macarena; Naranjo, Silvia; de la Calle-Mustienes, Elisa; Maeso, Ignacio; Beccari, Leonardo; Aneas, Ivy; Vielmas, Erika; Bovolenta, Paola; Nobrega, Marcelo A; Carvajal, Jaime; Gómez-Skarmeta, José Luis

    2015-06-16

    Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution. PMID:26034287

  10. Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

    PubMed Central

    Gómez-Marín, Carlos; Tena, Juan J.; Acemel, Rafael D.; López-Mayorga, Macarena; Naranjo, Silvia; de la Calle-Mustienes, Elisa; Maeso, Ignacio; Beccari, Leonardo; Aneas, Ivy; Vielmas, Erika; Bovolenta, Paola; Nobrega, Marcelo A.; Carvajal, Jaime; Gómez-Skarmeta, José Luis

    2015-01-01

    Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution. PMID:26034287

  11. 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.

  12. Comparison Of Multi-Frequency SAR Land Cover Signatures For Multi-Site Semi-Arid Regions Of Africa

    NASA Astrophysics Data System (ADS)

    Spies, Bernard; Lamb, Alistair; Brown, Sarah, Balzter, Heiko; Fisher, Peter

    2013-12-01

    This study shows the analysis and comparison of different SAR backscatter signatures (σ0 distributions) for distinguishable land cover types over two semi-arid test sites in Africa. The two sites that were chosen are located in Tanzania and Chad, where existing multi- frequency data was available from the different synthetic aperture radar (SAR) archives. Images were grouped into wet and dry season for the Tanzania site, whereas only dry season imagery was available for the Chad site. An IsoData unsupervised classification was applied on all three sets of images to classify seven land cover classes. Random samples were taken from each of the classes, resulting in σ0 distributions for the different classes for each site. These SAR land cover signatures are interpreted and discussed, with further steps identified.

  13. Mutation at a Strictly Conserved, Active Site Tyrosine in the Copper Amine Oxidase Leads to Uncontrolled Oxygenase Activity

    SciTech Connect

    Chen, Zhi-wei; Datta, Saumen; DuBois, Jennifer L.; Klinman, Judith P.; Mathews, F. Scott

    2010-09-07

    The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

  14. A New Quiet GSN Site at the South Pole: Comparison of Seismic Data Between SPA and QSPA.

    NASA Astrophysics Data System (ADS)

    Anderson, K. R.; Aster, R. C.; Butler, R.; Hutt, C.; Storm, T.; Anderson, D.; Vineyard, J. J.; Albert, D. G.

    2003-12-01

    Due to increasing noise from Amundsen-Scott South Pole Station (ASSPS), a new Global Seismic Network (GSN) station, QSPA, was constructed in the 2002-2003 field season to supplant the previous GSN station, SPA, in operation since c. 1985. QSPA is the first experiment established at the South Pole Remote Earth Science and Seismological Observatory (SPRESSO). The new instruments reside in the newly-designated seismic Quiet Sector, 8 km southeast of ASSPS. SPRESSO provides a much lower noise environment, yet is close enough to the south pole to be effectively co-sited with Earth's rotational axis for long-period seismological purposes. The SPRESSO site was selected after examining tractor surface noise propagation, balanced against power and communication considerations. Numerical modeling showed that ASSPS noise should be fairly well trapped in near-surface, lower-velocity firn zone and indicated that a burial depth of 300 m would very substantially reduce noise. QSPA consists of a Geotech Instruments KS-54000 at 275 m and Guralp CMG-3Tb at 255 m depth, separated horizontally by 10 m. These depths are approximately 160 m below the local firn/ice transition. QSPA also includes a near-surface vault ( ˜5 m depth) housing Streckeisen STS-1V and STS-2 sensors. SPA has been left in operation for a period of ˜1 yr to allow for a thorough comparison with QSPA. Although 8 km from the South Pole station activities, the QSPA site borehole instruments still sense South Pole noise. However, these noise levels are significantly diminished at frequencies >1 Hz and at periods >20 s. From 1 to 15 Hz, the improved noise environment is especially dramatic. For example, background noise at 2 Hz is 15 db below SPA, ˜20-25 dB at 3 Hz, ˜30-35 dB at 4Hz, and ˜35-40 dB above 5Hz. Between 2 and 10 Hz the QSPA Guralp borehole sensor shows data intervals with noise levels below the Peterson (1993) Low Noise Model (PLNM) (with minimum noise at ˜3Hz ˜12dB below PLNM), making the site among

  15. A single mutation in the hepta-peptide active site of Aspergillus niger PhyA phytase leads to myriad of biochemical changes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The active site motif of proteins belonging to ‘Histidine Acid Phosphatase’ (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs has revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger phyA phytase. However,...

  16. 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

  17. An ionizable active-site tryptophan imparts catalase activity to a peroxidase core.

    PubMed

    Loewen, Peter C; Carpena, Xavi; Vidossich, Pietro; Fita, Ignacio; Rovira, Carme

    2014-05-21

    Catalase peroxidases (KatG's) are bifunctional heme proteins that can disproportionate hydrogen peroxide (catalatic reaction) despite their structural dissimilarity with monofunctional catalases. Using X-ray crystallography and QM/MM calculations, we demonstrate that the catalatic reaction of KatG's involves deprotonation of the active-site Trp, which plays a role similar to that of the distal His in monofunctional catalases. The interaction of a nearby mobile arginine with the distal Met-Tyr-Trp essential adduct (in/out) acts as an electronic switch, triggering deprotonation of the adduct Trp.

  18. Comparison of Grab, Air, and Surface Results for Radiation Site Characterization

    NASA Astrophysics Data System (ADS)

    Glassford, Eric Keith

    2011-12-01

    energy photons from 234Th and 234mPa, the short-lived decay products of 238U, and 235U. Two sided t-tests and coefficient of variation were used to compare sampling types. The large grab samples had the lowest calculated coefficient of variation results for activity and atom percentage. The wipe samples had the highest calculated coefficient of variation of mean specific activity (dis/sec/g) for all three energies. The air filter samples had the highest coefficient of variation calculation for mean atom percentage, for both uranium isotopes examined. The data indicated that the large mass sample was the most effective at characterizing the rolling mill radioactive site conditions, since this would indicate which samples had the smallest variations compared to the mean. Additionally, measurement results of natural uranium in the samples indicate that the distribution of radioactive contamination at the sampling location is most likely non-homogeneous and that the size of the sample collected and analyzed must be sufficiently large to insure that the analytical results are truly representative of the activity present.

  19. Standardizing Clinical Trials Workflow Representation in UML for International Site Comparison

    PubMed Central

    de Carvalho, Elias Cesar Araujo; Jayanti, Madhav Kishore; Batilana, Adelia Portero; Kozan, Andreia M. O.; Rodrigues, Maria J.; Shah, Jatin; Loures, Marco R.; Patil, Sunita; Payne, Philip; Pietrobon, Ricardo

    2010-01-01

    Background With the globalization of clinical trials, a growing emphasis has been placed on the standardization of the workflow in order to ensure the reproducibility and reliability of the overall trial. Despite the importance of workflow evaluation, to our knowledge no previous studies have attempted to adapt existing modeling languages to standardize the representation of clinical trials. Unified Modeling Language (UML) is a computational language that can be used to model operational workflow, and a UML profile can be developed to standardize UML models within a given domain. This paper's objective is to develop a UML profile to extend the UML Activity Diagram schema into the clinical trials domain, defining a standard representation for clinical trial workflow diagrams in UML. Methods Two Brazilian clinical trial sites in rheumatology and oncology were examined to model their workflow and collect time-motion data. UML modeling was conducted in Eclipse, and a UML profile was developed to incorporate information used in discrete event simulation software. Results Ethnographic observation revealed bottlenecks in workflow: these included tasks requiring full commitment of CRCs, transferring notes from paper to computers, deviations from standard operating procedures, and conflicts between different IT systems. Time-motion analysis revealed that nurses' activities took up the most time in the workflow and contained a high frequency of shorter duration activities. Administrative assistants performed more activities near the beginning and end of the workflow. Overall, clinical trial tasks had a greater frequency than clinic routines or other general activities. Conclusions This paper describes a method for modeling clinical trial workflow in UML and standardizing these workflow diagrams through a UML profile. In the increasingly global environment of clinical trials, the standardization of workflow modeling is a necessary precursor to conducting a comparative

  20. 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)

  1. Refining the active site structure of iron-iron hydrogenase using computational infrared spectroscopy.

    PubMed

    Tye, Jesse W; Darensbourg, Marcetta Y; Hall, Michael B

    2008-04-01

    Iron-iron hydrogenases ([FeFe]H2ases) are exceptional natural catalysts for the reduction of protons to dihydrogen. Future biotechnological applications based on these enzymes require a precise understanding of their structures and properties. Although the [FeFe]H2ases have been characterized by single-crystal X-ray crystallography and a range of spectroscopic techniques, ambiguities remain regarding the details of the molecular structures of the spectroscopically observed forms. We use density functional theory (DFT) computations on small-molecule computational models of the [FeFe]H2ase active site to address this problem. Specifically, a series of structural candidates are geometry optimized and their infrared (IR) spectra are simulated using the computed C-O and C-N stretching frequencies and infrared intensities. Structural assignments are made by comparing these spectra to the experimentally determined IR spectra for each form. The H red form is assigned as a mixture of an Fe(I)Fe(I) form with an open site on the distal iron center and either a Fe(I)Fe(I) form in which the distal cyanide has been protonated or a Fe(II)Fe(II) form with a bridging hydride ligand. The Hox form is assigned as a valence-localized Fe(I)Fe(II) redox level with an open site at the distal iron. The Hox(air)(ox) form is assigned as an Fe(II)Fe(II) redox level with OH(-) or OOH(-) bound to the distal iron center that may or may not have an oxygen atom bound to one of the sulfur atoms of the dithiolate linker. Comparisons of the computed IR spectra of the (12)CO and (13)CO inhibited form with the experimental IR spectra show that exogenous CO binds terminally to the distal iron center.

  2. 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.

  3. Comparison between the accumulation capacity of four lichen species transplanted to a urban site.

    PubMed

    Bergamaschi, L; Rizzio, E; Giaveri, G; Loppi, S; Gallorini, M

    2007-07-01

    The capacity to accumulate trace elements from the atmosphere of the lichens Hypogymnia physodes, Parmelia sulcata, Pseudevernia furfuracea and Usnea gr. hirta transplanted to an urban site of N Italy was compared. Twenty-nine elements (Al, As, Br, Ca, Cd, Ce, Cl, Co, Cr, Cs, Cu, Fe, Hg, I, K, La, Mg, Mn, Ni, Pb, Rb, Sb, Sc, Se, Sm, Th, Ti, V, Zn) were analyzed by Instrumental Neutron Activation Analysis (INAA) and Electro-Thermal Atomic Absorption Spectroscopy (ET-AAS). The ratio between the concentrations of each element in exposed samples to that of control samples (exposed-to-control ratio and EC ratio) was used to investigate the accumulation rates of lichen thalli. The results showed that in general elements did not exhibit well defined trends, but rather showed fluctuations, and indicated that H. physodes, P. furfuracea and U. gr. hirta have a similar accumulation capacity, while that of P. sulcata is lower.

  4. 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

  5. A list of the most popular smoking cessation web sites and a comparison of their quality.

    PubMed

    Etter, Jean-François

    2006-12-01

    To identify the most popular smoking cessation Web sites and to compare their quality, we conducted an Internet survey of 706 U.S. current and former smokers who had already visited such a Web site. Participants cited 133 different sites and rated their quality. Surprisingly, two of the three most frequently visited Web sites were owned by tobacco companies: Philipmorrisusa.com and its information resource Quit Assist (cited by 15.9% of participants) and the R. J. Reynolds site (5.9%). However, these two sites were not perceived as helpful. The other most frequently visited sites were QuitNet.com (10.1% of all participants), the site of the American Lung Association (5.2%), and Webmd.com (4.4%). All the nine most frequently visited sites, which together attracted 56% of all visitors, ranked at or below average for quality or helpfulness, and together, the three sites with the highest quality ratings received only 7% of all visitors. Only one-third of participants received follow-up e-mails. Thirteen of the 18 most popular Web sites were for-profit, and several sites promoted unproven methods. Two sites ranked above average for quality, and both were nonprofit: Smokefree.gov and Anti-smoking.org. Quitsmoking.About.com ranked above average for helpfulness. Direct competition radically distinguishes Web sites from other smoking cessation services. However, competition resulted in the selection of tobacco companies sites and commercial sites rather than sites of the best quality. In general, users were not very satisfied with smoking cessation Web sites, and they had difficulty finding the best Web sites. Resources were scattered over dozens of Web sites of often mediocre quality that duplicated each other.

  6. Using catalytic atom maps to predict the catalytic functions present in enzyme active sites.

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-09-18

    Catalytic atom maps (CAMs) are minimal models of enzyme active sites. The structures in the Protein Data Bank (PDB) were examined to determine if proteins with CAM-like geometries in their active sites all share the same catalytic function. We combined the CAM-based search protocol with a filter based on the weighted contact number (WCN) of the catalytic residues, a measure of the "crowdedness" of the microenvironment around a protein residue. Using this technique, a CAM based on the Ser-His-Asp catalytic triad of trypsin was able to correctly identify catalytic triads in other enzymes within 0.5 Å rmsd of the CAM with 96% accuracy. A CAM based on the Cys-Arg-(Asp/Glu) active site residues from the tyrosine phosphatase active site achieved 89% accuracy in identifying this type of catalytic functionality. Both of these CAMs were able to identify active sites across different fold types. Finally, the PDB was searched to locate proteins with catalytic functionality similar to that present in the active site of orotidine 5'-monophosphate decarboxylase (ODCase), whose mechanism is not known with certainty. A CAM, based on the conserved Lys-Asp-Lys-Asp tetrad in the ODCase active site, was used to search the PDB for enzymes with similar active sites. The ODCase active site has a geometry similar to that of Schiff base-forming Class I aldolases, with lowest aldolase rmsd to the ODCase CAM at 0.48 Å. The similarity between this CAM and the aldolase active site suggests that ODCase has the correct catalytic functionality present in its active site for the generation of a nucleophilic lysine. PMID:22909276

  7. Using Catalytic Atom Maps to Predict the Catalytic Functions Present in Enzyme Active Sites

    PubMed Central

    Nosrati, Geoffrey R.; Houk, K. N.

    2012-01-01

    Catalytic Atom Maps (CAMs) are minimal models of enzyme active sites. The structures in the Protein Data Bank (PDB) were examined to determine if proteins with CAM-like geometries in their active sites all share the same catalytic function. We combined the CAM-based search protocol with a filter based on the weighted contact number (WCN) of the catalytic residues, a measure of the “crowdedness” of the microenvironment around a protein residue. Using this technique, a CAM based on the Ser-His-Asp catalytic triad of trypsin was able to correctly identify catalytic triads in other enzymes within 0.5 Å RMSD of the Catalytic Atom Map with 96% accuracy. A CAM based on the Cys-Arg-(Asp/Glu) active site residues from the tyrosine phosphatase active site achieved 89% accuracy in identifying this type of catalytic functionality. Both of these Catalytic Atom Maps were able to identify active sites across different fold types. Finally, the PDB was searched to locate proteins with catalytic functionality similar to that present in the active site of orotidine 5′-monophosphate decarboxylase (ODCase), whose mechanism is not known with certainty. A CAM, based on the conserved Lys-Asp-Lys-Asp tetrad in the ODCase active site, was used to search the PDB for enzymes with similar active sites. The ODCase active site has a geometry similar to that of Schiff base-forming Class I aldolases, with lowest aldolase RMSD to the ODCase CAM at 0.48 Å. The similarity between this CAM and the aldolase active site suggests that ODCase has the correct catalytic functionality present in its active site for the generation of a nucleophilic lysine. PMID:22909276

  8. Quantitative comparisons of in vitro assays for estrogenic activities.

    PubMed Central

    Fang, H; Tong, W; Perkins, R; Soto, A M; Prechtl, N V; Sheehan, D M

    2000-01-01

    Substances that may act as estrogens show a broad chemical structural diversity. To thoroughly address the question of possible adverse estrogenic effects, reliable methods are needed to detect and identify the chemicals of these diverse structural classes. We compared three assays--in vitro estrogen receptor competitive binding assays (ER binding assays), yeast-based reporter gene assays (yeast assays), and the MCF-7 cell proliferation assay (E-SCREEN assay)--to determine their quantitative agreement in identifying structurally diverse estrogens. We examined assay performance for relative sensitivity, detection of active/inactive chemicals, and estrogen/antiestrogen activities. In this examination, we combined individual data sets in a specific, quantitative data mining exercise. Data sets for at least 29 chemicals from five laboratories were analyzed pair-wise by X-Y plots. The ER binding assay was a good predictor for the other two assay results when the antiestrogens were excluded (r(2) is 0.78 for the yeast assays and 0.85 for the E-SCREEN assays). Additionally, the examination strongly suggests that biologic information that is not apparent from any of the individual assays can be discovered by quantitative pair-wise comparisons among assays. Antiestrogens are identified as outliers in the ER binding/yeast assay, while complete antagonists are identified in the ER binding and E-SCREEN assays. Furthermore, the presence of outliers may be explained by different mechanisms that induce an endocrine response, different impurities in different batches of chemicals, different species sensitivity, or limitations of the assay techniques. Although these assays involve different levels of biologic complexity, the major conclusion is that they generally provided consistent information in quantitatively determining estrogenic activity for the five data sets examined. The results should provide guidance for expanded data mining examinations and the selection of appropriate

  9. 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.

  10. 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

  11. Parameterization 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.

    2012-12-01

    Thermokarst features are thought to be an important mechanism for landscape change in permafrost-dominated cold regions, but few such features have been incorporated into full featured landscape models. The root of this shortcoming is that historic observations are not detailed enough to parameterize a model, and the models typically do not include the relevant processes for thermal erosion. A new, dynamic thermokarst feature has been identified at the Caribou-Poker Creek Research Watershed (CPCRW) in the boreal forest of Interior Alaska. Located adjacent to a traditional use trail, this feature terminates directly in Caribou Creek. Erosion within the feature is driven predominantly by fluvial interflow. CPCRW is a Long-Term Ecological Research site underlain by varying degrees of relatively warm, discontinuous permafrost. This poster will describe the suite of measurements that have been undertaken to parameterize the ERODE model for this site, including thorough surveys, time lapse- and aerial photography, and 3-D structure from motion algorithms.

  12. 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…

  13. Corrective Action Site 02-37-02 Background Information and Comparison to Corrective Action Site 09-99-06

    SciTech Connect

    2012-06-26

    Corrective Action Site (CAS) 02-37-02, Gas Sampling Assembly, is associated with nuclear test MULLET. MULLET was an underground safety test conducted on October 17, 1963. The experiment also involved prompt sampling of particulate material from the detonation, similar to CAS 09-99-06, Gas Sampling Assembly, which is associated with PLAYER/YORK. The sampling system at MULLET was similar to that of PLAYER/YORK and was used to convey gas from the MULLET emplacement hole (U2ag) to a sampling assembly. Beyond the sampling assembly, the system had a 'Y' junction with one branch running to a filter unit and the other running to a scrubber unit. The total system length was approximately 250 feet and is depicted on the attached drawing. According to the available background information, retrieval of the sample material from the MULLET event caused significant alpha (plutonium) contamination, limited to an area near ground zero (GZ). Test support Radiological Control Technicians did not detect contamination outside the immediate GZ area. In addition, vehicles, equipment, and workers that were contaminated were decontaminated on site. Soil contamination was addressed through the application of oil, and the site was decommissioned after the test. Any equipment that could be successfully decontaminated and had a future use was removed from the site. The contaminated equipment and temporary buildings erected to support the test were buried on site, most likely in the area under the dirt berm. The exact location of the buried equipment and temporary buildings is unknown. No information was found describing the disposition of the filter and scrubber, but they are not known to be at the site. The COMMODORE test was conducted at U2am on May 20, 1967, and formed the crater next to CAS 02-37-02. The COMMODORE test area had been surveyed prior to the test, and alpha contamination was not identified. Furthermore, alpha contamination was not identified during the COMMODORE re

  14. Pesticides analysed in rainwater in Alsace region (Eastern France): Comparison between urban and rural sites

    NASA Astrophysics Data System (ADS)

    Scheyer, Anne; Morville, Stéphane; Mirabel, Philippe; Millet, Maurice

    during this period. This is the case for diflufenican which was detected only during application. Two important peaks of concentrations were observed; a first one (101 ng L -1) in Erstein in November 2002 (4-11 November) and a second one (762 ng L -1) also in Erstein (28 April-15 May). The same behaviour can be seen for chlorfenvinphos and phosalone which have been detected, respectively, 2 and 4 times in Erstein and Strasbourg at high concentrations (28 April 2003-15 May 2003, 187 ng L -1 of phosalone and 157 ng L -1 of chlorfenvinphos in Erstein). MCPP, 2,4 MCPA and 2,4-D have been detected at high concentrations in rainwater but for the other pesticides very episodically and mainly during their use in agriculture. Maximal concentrations of MCPP and 2,4 MCPA have been measured in Erstein between 28 April and 15 May (904 and 746 ng L -1, respectively). Comparison between rural and urban sites showed that concentrations in rural areas are generally higher except for pesticides commonly applied in urban areas like Diuron. No seasonal phenomenon was observed for Diuron. This herbicide has been detected in practically all of the rainwater samples in Strasbourg (40/41) with a maximum of 1025 ng L -1 (16-23 September 2002) in 38 samples on 41 in Erstein with a maximum of 317 ng L -1 (15-23 October 2002). The total concentration of Diuron measured between 4 March 2002 and 20 July 2003 is of 4721 ng L -1 in Strasbourg and 5025 ng L -1 in Erstein. This result shows that wet deposition of Diuron in urban and rural sites was equivalent and can be explained by the "urban use" of this molecule together with its potential persistence.

  15. 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.

  16. 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.

  17. Identification of ice nucleation active sites on feldspar dust particles.

    PubMed

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

    2015-03-19

    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

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

    PubMed Central

    2015-01-01

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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. Amyloid-β peptide active site: theoretical Cu K-edge XANES study

    NASA Astrophysics Data System (ADS)

    Chaynikov, A. P.; Soldatov, M. A.; Streltsov, V.; Soldatov, A. V.

    2013-04-01

    This article is dedicated to the local atomic structure analysis of the copper binding site in amyloid-β peptide. Here we considered two possible structural models that were previously obtained by means of EXAFS analysis and density functional theory simulations. We present the calculations of Cu K-edge XANES spectra for both models and make comparison of these spectra with experiment.

  4. A Comparison between Lightning Activity and Passive Microwave Measurements

    NASA Technical Reports Server (NTRS)

    Kevin, Driscoll T.; Hugh, Christian J.; Goodman, Steven J.

    1999-01-01

    A recent examination of data from the Lightning Imaging Sensor (LIS) and the TRMM Microwave Imager (TMI) suggests that storm with the highest frequency of lightning also possess the most pronounced microwave scattering signatures at 37 and 85 GHz. This study demonstrates a clear dependence between lightning and the passive microwave measurements, and accentuates how direct the relationship really is between cloud ice and lightning activity. In addition, the relationship between the quantity of ice content and the frequency of lightning (not just the presence of lightning) , is consistent throughout the seasons in a variety of regimes. Scatter plots will be presented which show the storm-averaged brightness temperatures as a function of the lightning density of the storms (L/Area) . In the 85 GHz and 37 GHz scatter plots, the brightness temperature is presented in the form Tb = k1 x log10(L/Area) + k2, where the slope of the regression, k1, is 58 for the 85 GHz relationship and 30.7 for the 37 GHz relationship. The regression for both these fits showed a correlation of 0.76 (r2 = 0.58), which is quite promising considering the simple procedure used to make the comparisons, which have not yet even been corrected for the view angle differences between the instruments, or the polarization corrections in the microwave imager.

  5. Wind load comparison for the ASCE standard 7 and the Hanford site design criteria

    SciTech Connect

    Giller, R.A., Westinghouse Hanford

    1996-07-16

    This document provides calculations and discussions to compare Hanford Site wind load criteria with the current national standard for wind loads (ASCE 7, 1995). Site criteria uses the 1988 edition ASCE 7.

  6. Possible active site of the sweet-tasting protein thaumatin.

    PubMed

    Slootstra, J W; De Geus, P; Haas, H; Verrips, C T; Meloen, R H

    1995-10-01

    Epitopes on thaumatin and monellin were studied using the PEPSCAN-technology. The antibodies used were raised against thaumatin. Only antibodies that, in an ELISA, both recognized thaumatin and monellin were used in the PEPSCAN-analyses. On thaumatin two major overlapping epitopes were identified. On monellin no epitopes could be identified. The identified epitope region on thaumatin shares structural features with various peptide and protein sweeteners. It contains an aspartame-like site which is formed by Asp21 and Phe80, tips of the two extruding loops KGDAALDAGGR19-29 and CKRFGRPP77-84, which are spatially positioned next to each other. Furthermore, sub-sequences of the KGDAALDAGGR19-29 loop are similar to peptide-sweeteners such as L-Asp-D-Ala-L-Ala-methyl ester and L-Asp-D-Ala-Gly-methyl ester. Since the aspartame-like Asp21-Phe80 site and the peptide-sweetener-like sequences are also not present in non-sweet thaumatin-like proteins it is postulated that the KGDAALDAGGR19-29- and CKRFGRPP77-84 loop contain important sweet-taste determinants. This region has previously not been implicated as a sweet-taste determinant of thaumatin.

  7. A comparison of 4 methods of data presentation for lysosomal enzyme activity in gingival crevicular fluid.

    PubMed

    Lamster, I B; Oshrain, R L; Fiorello, L A; Celenti, R S; Gordon, J M

    1988-07-01

    In previous studies, we have emphasized the importance of considering the methods used for analysis of gingival crevicular fluid (GCF). This study evaluated 4 different approaches for data presentation of lysosomal enzyme activity in GCF. GCF was collected from patients displaying at least 2 mm of clinical attachment loss at a minimum of 3 sites in the mouth (DA), and patients who did not display clinical attachment loss of 2 mm or more at any site in the mouth (DI), during a 3-month interval following entry into a longitudinal trial. GCF was collected by the timed intrasulcular placement of precut filter paper strips. 16 to 28 individual GCF samples were collected from each patient. The lysosomal enzymes studied were B-glucuronidase (BG) and arylsulfatase. The mean values for the DA and DI groups at baseline and 3 months are reported. The results indicate that when the data is expressed as total enzyme activity (unit activity) per 30-s collection (UA) or UA x GCF volume (microliter) per mm of probing depth, the DA group demonstrated significantly greater mean values than the DI group at baseline and 3 months. In contrast, when the data was expressed as concentration (UA/microliter), or UA per mm of probing depth, differences between the DA and DI groups were observed only at the 3-month evaluation. The difficulty in using concentration when reporting GCF lysosomal enzyme activity is emphsized by comparison of the data from the DA group and the high and low enzyme activity subsets of the DI group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3042812

  8. 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.

  9. 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

  10. A comparison of the photocatalytic activity of six tunneled titanates

    SciTech Connect

    Sanford, Stephen; Misture, Scott T.; Edwards, Doreen D.

    2013-04-15

    The photocatalytic behavior of six tunneled titanates—Na{sub 0.7}Ga{sub 4.7}Ti{sub 0.3}O{sub 8}, Na{sub 0.8}Ga{sub 4.8}Ti{sub 1.2}O{sub 10}, Na{sub 0.8}Ga{sub 4.8}Ti{sub 2.2}O{sub 12}, K{sub 1}Ga{sub 17}Ti{sub 15}O{sub 56}, K{sub 1.5}Ga{sub 1.5}Ti{sub 6.5}O{sub 16}, and BaTi{sub 4}O{sub 9}—was investigated using methylene blue as an indicator and a xenon arc lamp as the radiation source. Powders prepared by solid state reaction had surface areas ranging from 0.40 to 1.58 m{sup 2}/g and particle sizes ranging from 1 to 25 μm. Bandgaps, as measured from diffuse reflectance data, ranged from 2.84 to 4.15 eV. Two of the tunneled titanates—Na{sub 0.8}Ga{sub 4.8}Ti{sub 1.2}O{sub 10} and Na{sub 0.7}Ga{sub 4.7}Ti{sub 0.3}O{sub 8}—exhibited negligible photocatalytic activity. The activities of Na{sub 0.8}Ga{sub 4.8}Ti{sub 2.2}O{sub 12}, KGa{sub 17}Ti{sub 15}O{sub 56}, and K{sub 1.5}Ga{sub 1.5}Ti{sub 6.5}O{sub 16} were similar to each other, exhibiting apparent first-order reaction rates of 0.275 to 0.31 h{sup −1} using 100 mg of powder in 125 ml of 20 μM methylene blue at room temperature. The BaTi{sub 4}O{sub 9} sample exhibited the highest photocatalytic activity with an apparent first-order reaction rate of 0.53 h{sup −1} under the same conditions. The addition of a RuO{sub 2} co-catalyst improved the photocatalytic activity of Na{sub 0.7}Ga{sub 4.7}Ti{sub 0.3}O{sub 8}, decreased the activity of BaTi{sub 4}O{sub 9}, and had little effect on the activity of the other powders. A comparison of the structural features in the six materials shows that photocatalytic activity is strongly related to the density of TiO{sub 6} octahedra in the different crystal structures. Among the gallium-containing tunneled titanates, photocatalytic activity increased with decreasing band gap. - Graphical abstract: Na{sub 0.7}Ga{sub 4.7}Ti{sub 0.3}O{sub 8}—One of the tunneled titanates investigated. Highlights: ► The photocatalytic activity of six tunneled titanates was

  11. 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.

  12. 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.

  13. 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.

  14. Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1

    PubMed Central

    Arena de Souza, Victoria; Scott, David J.; Nettleship, Joanne E.; Rahman, Nahid; Charlton, Michael H.; Walsh, Martin A.; Owens, Raymond J.

    2015-01-01

    Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme. PMID:26657071

  15. A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations.

    PubMed

    O'Farrell, Norah; Kreiner, Michaela; Moore, Barry D; Parker, Marie-Claire

    2006-11-01

    We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).

  16. 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.

  17. Mechanistic pathways of mercury removal from the organomercurial lyase active site.

    PubMed

    Silva, Pedro J; Rodrigues, Viviana

    2015-01-01

    protonation by solvent-provided H3O(+). Thiolate addition to the active site (prior to any attack by thiols) leads to pathways where the removal of the first cysteine becomes the rate-determining step, irrespective of whether Cys159 or Cys96 leaves first. Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction. Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket. PMID:26246970

  18. Mechanistic pathways of mercury removal from the organomercurial lyase active site

    PubMed Central

    Rodrigues, Viviana

    2015-01-01

    protonation by solvent-provided H3O+. Thiolate addition to the active site (prior to any attack by thiols) leads to pathways where the removal of the first cysteine becomes the rate-determining step, irrespective of whether Cys159 or Cys96 leaves first. Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction. Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket. PMID:26246970

  19. 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…

  20. Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity.

    PubMed

    Bright, Nicholas A; Davis, Luther J; Luzio, J Paul

    2016-09-12

    The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid hydrolase active. We found that endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid-hydrolase-active endolysosomes and acid-hydrolase-inactive, terminal storage lysosomes exist in dynamic equilibrium. We conclude that the terminal endocytic compartment is composed of acid-hydrolase-active, acidic endolysosomes and acid hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle. PMID:27498570

  1. Surgical site infection after caesarean section: space for post-discharge surveillance improvements and reliable comparisons.

    PubMed

    Ferraro, Federica; Piselli, Pierluca; Pittalis, Silvia; Ruscitti, Luca E; Cimaglia, Claudia; Ippolito, Giuseppe; Puro, Vincenzo

    2016-04-01

    Surgical site infections (SSI) after caesarean section (CS) represent a substantial health system concern. Surveying SSI has been associated with a reduction in SSI incidence. We report the findings of three (2008, 2011 and 2013) regional active SSI surveillances after CS in community hospital of the Latium region determining the incidence of SSI. Each CS was surveyed for SSI occurrence by trained staff up to 30 post-operative days, and association of SSI with relevant characteristics was assessed using binomial logistic regression. A total of 3,685 CS were included in the study. A complete 30 day post-operation follow-up was achieved in over 94% of procedures. Overall 145 SSI were observed (3.9% cumulative incidence) of which 131 (90.3%) were superficial and 14 (9.7%) complex (deep or organ/space) SSI; overall 129 SSI (of which 89.9% superficial) were diagnosed post-discharge. Only higher NNIS score was significantly associated with SSI occurrence in the regression analysis. Our work provides the first regional data on CS-associated SSI incidence, highlighting the need for a post-discharge surveillance which should assure 30 days post-operation to not miss data on complex SSI, as well as being less labour intensive.

  2. Surgical site infection after caesarean section: space for post-discharge surveillance improvements and reliable comparisons.

    PubMed

    Ferraro, Federica; Piselli, Pierluca; Pittalis, Silvia; Ruscitti, Luca E; Cimaglia, Claudia; Ippolito, Giuseppe; Puro, Vincenzo

    2016-04-01

    Surgical site infections (SSI) after caesarean section (CS) represent a substantial health system concern. Surveying SSI has been associated with a reduction in SSI incidence. We report the findings of three (2008, 2011 and 2013) regional active SSI surveillances after CS in community hospital of the Latium region determining the incidence of SSI. Each CS was surveyed for SSI occurrence by trained staff up to 30 post-operative days, and association of SSI with relevant characteristics was assessed using binomial logistic regression. A total of 3,685 CS were included in the study. A complete 30 day post-operation follow-up was achieved in over 94% of procedures. Overall 145 SSI were observed (3.9% cumulative incidence) of which 131 (90.3%) were superficial and 14 (9.7%) complex (deep or organ/space) SSI; overall 129 SSI (of which 89.9% superficial) were diagnosed post-discharge. Only higher NNIS score was significantly associated with SSI occurrence in the regression analysis. Our work provides the first regional data on CS-associated SSI incidence, highlighting the need for a post-discharge surveillance which should assure 30 days post-operation to not miss data on complex SSI, as well as being less labour intensive. PMID:27196552

  3. Outside-binding site mutations modify the active site's shapes in neuraminidase from influenza A H1N1.

    PubMed

    Tolentino-Lopez, Luis; Segura-Cabrera, Aldo; Reyes-Loyola, Paola; Zimic, Mirko; Quiliano, Miguel; Briz, Veronica; Muñoz-Fernández, Angeles; Rodríguez-Pérez, Mario; Ilizaliturri-Flores, Ian; Correa-Basurto, Jose

    2013-01-01

    The recent occurrence of 2009 influenza A (H1N1) pandemic as well as others has raised concern of a far more dangerous outcome should this virus becomes resistant to current drug therapies. The number of clinical cases that are resistant to oseltamivir (Tamiflu®) is larger than the limited number of neuraminidase (NA) mutations (H275Y, N295S, and I223R) that have been identified at the active site and that are associated to oseltamivir resistance. In this study, we have performed a comparative analysis between a set of NAs that have the most representative mutations located outside the active site. The recently crystallized NA-oseltamivir complex (PDB ID: 3NSS) was used as a wild-type structure. After selecting the target NA sequences, their three-dimensional (3D) structure was built using 3NSS as a template by homology modeling. The 3D NA models were refined by molecular dynamics (MD) simulations. The refined models were used to perform a docking study, using oseltamivir as a ligand. Furthermore, the docking results were refined by free-energy analysis using the MM-PBSA method. The analysis of the MD simulation results showed that the NA models reached convergence during the first 10 ns. Visual inspection and structural measures showed that the mutated NA active sites show structural variations. The docking and MM-PBSA results from the complexes showed different binding modes and free energy values. These results suggest that distant mutations located outside the active site of NA affect its structure and could be considered to be a new source of resistance to oseltamivir, which agrees with reports in the clinical literature.

  4. 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.

  5. Aerosol Light Absorption and Scattering at Four Sites in and Near Mexico City: Comparison with Las Vegas, Nevada, USA

    NASA Astrophysics Data System (ADS)

    Arnott, W. P.; Miranda, G. P.; Gaffney, J. S.; Marley, N. A.

    2007-05-01

    Four photoacoustic spectrometers (PAS) for aerosol light scattering and absorption measurements were deployed in and near Mexico City in March 2006 as part of the Megacity Impacts on Regional and Global Environments (MIRAGE). The four sites included: an urban site at Instituto Mexicano del Petroleo (Mexican Oil Institute, denoted by IMP); a suburban site at the Technological University of Tecamac; a rural site at "La Biznaga" ranch; and a site at the Paseo de Cortes (altitude 3,810 meters ASL) in the rural area above Amecameca in the State of Mexico, on the saddle between the volcanoes Popocatepetl and Iztaccihuatl. A similar campaign was held in Las Vegas, Nevada, USA in January-February, 2003. The IMP site gave in-situ characterization of the Mexico City plume under favorable wind conditions while the other sites provided characterization of the plume, mixed in with any local sources. The second and third sites are north of Mexico City, and the fourth site is south. The PAS used at IMP operates at 532 nm, and conveniently allowed for characterization of gaseous absorption at this wavelength as well. Instruments at the second and third sites operate at 870 nm, and the one at the fourth site at 780 nm. Light scattering measurements are accomplished within the PAS by the reciprocal nephelometery method. In the urban site the aerosol absorption coefficient typically varies between 20 and 180 Mm-1 during the course of the day and significant diurnal variation of the aerosol single scattering albedo was observed probably as a consequence of secondary aerosol formation. Comparisons with TSI nephelometer scattering at the T0 site will be presented. We will present the diurnal variation of the scattering and absorption as well as the single scattering albedo and fraction of absorption due to gases at the IMP site and compare with Las Vegas diurnal variation. Mexico City 'breaths' more during the course of the day than Las Vegas, Nevada in part because the latitude of

  6. Structural Analysis of an Open Active Site Conformation of Nonheme Iron Halogenase CytC3

    SciTech Connect

    Wong, Cintyu; Galonic Fujimori, Danica; Walsh, Christopher T.; Drennan, Catherine L.

    2009-04-28

    CytC3, a member of the recently discovered class of nonheme Fe(II) and {alpha}-ketoglutarate ({alpha}KG)-dependent halogenases, catalyzes the double chlorination of l-2-aminobutyric acid (Aba) to produce a known Streptomyces antibiotic, {gamma},{gamma}-dichloroaminobutyrate. Unlike the majority of the Fe(II)-{alpha}KG-dependent enzymes that catalyze hydroxylation reactions, halogenases catalyze a transfer of halides. To examine the important enzymatic features that discriminate between chlorination and hydroxylation, the crystal structures of CytC3 both with and without {alpha}KG/Fe(II) have been solved to 2.2 {angstrom} resolution. These structures capture CytC3 in an open active site conformation, in which no chloride is bound to iron. Comparison of the open conformation of CytC3 with the closed conformation of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound FeCl catalyst: (1) the presence of a hydrogen-bonding network between the chloride and surrounding residues, and (2) the presence of a hydrophobic pocket in which the chloride resides.

  7. 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

  8. Comparison of Atmospheric Deposition Among Three Sites In and Near the Flat Tops Wilderness Area, Colorado, 2003-2005

    USGS Publications Warehouse

    Ingersoll, George P.; Campbell, Donald H.; Mast, M. Alisa

    2008-01-01

    Atmospheric deposition was monitored for ammonium, nitrate, and sulfate concentrations and precipitation amounts in the Flat Tops Wilderness Area of northwestern Colorado at Ned Wilson Lake beginning in 1984 to detect changes that might result from future emissions associated with development of oil-shale resources in northwestern Colorado. Renewed monitoring, by the U.S. Geological Survey, in cooperation with Rio Blanco County, to determine the current status of atmospheric deposition has been ongoing since 2003 at Ned Wilson Lake. Two new monitoring sites were located near Ripple Creek Pass near the Flat Tops Wilderness area and about 12 kilometers north of Ned Wilson Lake because access to the area near Ripple Creek Pass is less difficult and less expensive, particularly in winter and spring. The intent of this study was to establish whether the new deposition data being collected near Ripple Creek Pass, near the northern boundary of the Flat Tops Wilderness Area, would be representative of deposition at sensitive sites within the wilderness such as Ned Wilson Lake and to compare more current (2003 through 2005) deposition data with earlier data (1984 through 1991). At Ned Wilson Lake, bulk ammonium and nitrate concentrations collected from 1984 through 1991 were similar to those from 2003 through 2005. However, in the same comparison significant differences in sulfate concentrations were observed, indicating a decrease consistent with other regional findings for similar periods. Comparison of concentrations of constituents at two bulk-deposition sites located at Ned Wilson Lake (NWLB) and near Ripple Creek Pass (RCPB) showed only one significant difference (p = 0.05) with the winter bulk nitrate concentrations for NWLB significantly lower than winter concentrations from RCPB. Another comparison of concentrations of constituents between the bulk deposition site RCPB and a wet deposition site 100 meters away (RCPW) showed no significant differences for

  9. Approximated maximum adsorption of His-tagged enzyme/mutants on Ni2+-NTA for comparison of specific activities.

    PubMed

    Li, Yuanli; Long, Gaobo; Yang, Xiaolan; Hu, Xiaolei; Feng, Yiran; Tan, Deng; Xie, Yanling; Pu, Jun; Liao, Fei

    2015-03-01

    By approximating maximum activities of six-histidine (6His)-tagged enzyme/mutants adsorbed on Ni2+-NTA-magnetic-submicron-particle (Ni2+-NTA-MSP), a facile approach was tested for comparing enzyme specific activities in cell lysates. On a fixed quantity of Ni2+-NTA-MSP, the activity of an adsorbed 6His-tagged enzyme/mutant was measured via spectrophotometry; the activity after saturation adsorption (Vs) was predicted from response curve with quantities of total proteins from the same lysate as the predictor; Vs was equivalent of specific activity for comparison. This approach required abundance of a 6His-tagged enzyme/mutant over 3% among total proteins in lysate, an accurate series of quantities of total proteins from the same lysate, the largest activity generated by enzyme occupying over 85% binding sites on Ni2+-NTA-MSP and the minimum activity as absorbance change rates of 0.003 min(-1) for analysis. The prediction of Vs tolerated errors in concentrations of total proteins in lysates and was effective to 6His-tagged alkaline phosphatase and its 6His-tagged mutant in lysates. Notably, of those two 6His-tagged enzymes, Vs was effectively approximated with just one optimized quantity of lysates. Hence, this approach with Ni2+-NTA-MSP worked for comparison of specific activities of 6His-tagged enzyme/mutants in lysates when they had sufficient abundance among proteins and activities of adsorbed enzymes were measurable.

  10. Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site.

    PubMed

    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

  11. 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

  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. Coordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases.

    PubMed

    Koutsioulis, Dimitris; Lyskowski, Andrzej; Mäki, Seija; Guthrie, Ellen; Feller, Georges; Bouriotis, Vassilis; Heikinheimo, Pirkko

    2010-01-01

    Alkaline phosphatases (APs) are commercially applied enzymes that catalyze the hydrolysis of phosphate monoesters by a reaction involving three active site metal ions. We have previously identified H135 as the key residue for controlling activity of the psychrophilic TAB5 AP (TAP). In this article, we describe three X-ray crystallographic structures on TAP variants H135E and H135D in complex with a variety of metal ions. The structural analysis is supported by thermodynamic and kinetic data. The AP catalysis essentially requires octahedral coordination in the M3 site, but stability is adjusted with the conformational freedom of the metal ion. Comparison with the mesophilic Escherichia coli, AP shows differences in the charge transfer network in providing the chemically optimal metal combination for catalysis. Our results provide explanation why the TAB5 and E. coli APs respond in an opposite way to mutagenesis in their active sites. They provide a lesson on chemical fine tuning and the importance of the second coordination sphere in defining metal specificity in enzymes. Understanding the framework of AP catalysis is essential in the efforts to design even more powerful tools for modern biotechnology. PMID:19916164

  14. A Mutational Analysis of the Active Site Loop Residues in cis-3-Chloroacrylic Acid Dehalogenase

    PubMed Central

    Schroeder, Gottfried K.; Huddleston, Jamison P.; Johnson, William H.; Whitman, Christian P.

    2013-01-01

    cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) from Pseudomonas pavonaceae 170 and a homologue from Corynebacterium glutamicum designated Cg10062 share 34% sequence identity (54% similarity). The former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. Although Cg10062 has the six active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114) that are critical for cis-CaaD activity, it shows only a low level cis-CaaD activity and lacks the specificity of cis-CaaD: Cg10062 processes both isomers of 3-chloroacrylate with a preference for the cis-isomer. Although the basis for these differences is unknown, a comparison of the crystal structures of the enzymes covalently modified by an adduct resulting from their incubation with the same inhibitor offers a possible explanation. A 6-residue active site loop in cis-CaaD shows a strikingly different conformation from that observed in Cg10062: the loop closes down on the active site of cis-CaaD, but not on that of Cg10062. In order to examine what this loop might contribute to cis-CaaD catalysis and specificity, the residues were changed individually to those found in Cg10062. Subsequent kinetic and mechanistic analysis suggests that the T34A mutant of cis-CaaD is more Cg10062-like. The mutant enzyme shows a 4-fold increase in Km (using cis-3-bromoacrylate), but not to the degree observed for Cg10062 (687-fold). The mutation also causes a 4-fold decrease in the burst rate (compared to the wild type cis-CaaD), whereas Cg10062 shows no burst rate. More telling is the reaction of the T34A mutant of cis-CaaD with the alternate substrate, 2,3-butadienoate. In the presence of NaBH4 and the allene, cis-CaaD is completely inactivated after one turnover due to the covalent modification of Pro-1. The same experiment with Cg10062 does not result in the covalent modification of Pro-1. The different outcomes are attributed to

  15. Electronic Cigarette Marketing Online: a Multi-Site, Multi-Product Comparison

    PubMed Central

    Sidhu, Anupreet K; Valente, Thomas W

    2015-01-01

    Background Electronic cigarette awareness and use has been increasing rapidly. E-cigarette brands have utilized social networking sites to promote their products, as the growth of the e-cigarette industry has paralleled that of Web 2.0. These online platforms are cost-effective and have unique technological features and user demographics that can be attractive for selective marketing. The popularity of multiple sites also poses a risk of exposure to social networks where e-cigarette brands might not have a presence. Objective To examine the marketing strategies of leading e-cigarette brands on multiple social networking sites, and to identify how affordances of the digital media are used to their advantage. Secondary analyses include determining if any brands are benefitting from site demographics, and exploring cross-site diffusion of marketing content through multi-site users. Methods We collected data from two e-cigarette brands from four social networking sites over approximately 2.5 years. Content analysis is used to search for themes, population targeting, marketing strategies, and cross-site spread of messages. Results Twitter appeared to be the most frequently used social networking site for interacting directly with product users. Facebook supported informational broadcasts, such as announcements regarding political legislation. E-cigarette brands also differed in their approaches to their users, from informal conversations to direct product marketing. Conclusions E-cigarette makers use different strategies to market their product and engage their users. There was no evidence of direct targeting of vulnerable populations, but the affordances of the different sites are exploited to best broadcast context-specific messages. We developed a viable method to study cross-site diffusion, although additional refinement is needed to account for how different types of digital media are used. PMID:27227129

  16. An Activity Index for Raw Accelerometry Data and Its Comparison with Other Activity Metrics.

    PubMed

    Bai, Jiawei; Di, Chongzhi; Xiao, Luo; Evenson, Kelly R; LaCroix, Andrea Z; Crainiceanu, Ciprian M; Buchner, David M

    2016-01-01

    Accelerometers have been widely deployed in public health studies in recent years. While they collect high-resolution acceleration signals (e.g., 10-100 Hz), research has mainly focused on summarized metrics provided by accelerometers manufactures, such as the activity count (AC) by ActiGraph or Actical. Such measures do not have a publicly available formula, lack a straightforward interpretation, and can vary by software implementation or hardware type. To address these problems, we propose the physical activity index (AI), a new metric for summarizing raw tri-axial accelerometry data. We compared this metric with the AC and another recently proposed metric for raw data, Euclidean Norm Minus One (ENMO), against energy expenditure. The comparison was conducted using data from the Objective Physical Activity and Cardiovascular Health Study, in which 194 women 60-91 years performed 9 lifestyle activities in the laboratory, wearing a tri-axial accelerometer (ActiGraph GT3X+) on the hip set to 30 Hz and an Oxycon portable calorimeter, to record both tri-axial acceleration time series (converted into AI, AC, and ENMO) and oxygen uptake during each activity (converted into metabolic equivalents (METs)) at the same time. Receiver operating characteristic analyses indicated that both AI and ENMO were more sensitive to moderate and vigorous physical activities than AC, while AI was more sensitive to sedentary and light activities than ENMO. AI had the highest coefficients of determination for METs (0.72) and was a better classifier of physical activity intensity than both AC (for all intensity levels) and ENMO (for sedentary and light intensity). The proposed AI provides a novel and transparent way to summarize densely sampled raw accelerometry data, and may serve as an alternative to AC. The AI's largely improved sensitivity on sedentary and light activities over AC and ENMO further demonstrate its advantage in studies with older adults. PMID:27513333

  17. An Activity Index for Raw Accelerometry Data and Its Comparison with Other Activity Metrics

    PubMed Central

    Bai, Jiawei; Di, Chongzhi; Xiao, Luo; Evenson, Kelly R.; LaCroix, Andrea Z.; Crainiceanu, Ciprian M.; Buchner, David M.

    2016-01-01

    Accelerometers have been widely deployed in public health studies in recent years. While they collect high-resolution acceleration signals (e.g., 10–100 Hz), research has mainly focused on summarized metrics provided by accelerometers manufactures, such as the activity count (AC) by ActiGraph or Actical. Such measures do not have a publicly available formula, lack a straightforward interpretation, and can vary by software implementation or hardware type. To address these problems, we propose the physical activity index (AI), a new metric for summarizing raw tri-axial accelerometry data. We compared this metric with the AC and another recently proposed metric for raw data, Euclidean Norm Minus One (ENMO), against energy expenditure. The comparison was conducted using data from the Objective Physical Activity and Cardiovascular Health Study, in which 194 women 60–91 years performed 9 lifestyle activities in the laboratory, wearing a tri-axial accelerometer (ActiGraph GT3X+) on the hip set to 30 Hz and an Oxycon portable calorimeter, to record both tri-axial acceleration time series (converted into AI, AC, and ENMO) and oxygen uptake during each activity (converted into metabolic equivalents (METs)) at the same time. Receiver operating characteristic analyses indicated that both AI and ENMO were more sensitive to moderate and vigorous physical activities than AC, while AI was more sensitive to sedentary and light activities than ENMO. AI had the highest coefficients of determination for METs (0.72) and was a better classifier of physical activity intensity than both AC (for all intensity levels) and ENMO (for sedentary and light intensity). The proposed AI provides a novel and transparent way to summarize densely sampled raw accelerometry data, and may serve as an alternative to AC. The AI’s largely improved sensitivity on sedentary and light activities over AC and ENMO further demonstrate its advantage in studies with older adults. PMID:27513333

  18. Spectroscopic definition of the copper active sites in mordenite: selective methane oxidation.

    PubMed

    Vanelderen, Pieter; Snyder, Benjamin E R; Tsai, Ming-Li; Hadt, Ryan G; Vancauwenbergh, Julie; Coussens, Olivier; Schoonheydt, Robert A; Sels, Bert F; Solomon, Edward I

    2015-05-20

    Two distinct [Cu-O-Cu](2+) sites with methane monooxygenase activity are identified in the zeolite Cu-MOR, emphasizing that this Cu-O-Cu active site geometry, having a ∠Cu-O-Cu ∼140°, is particularly formed and stabilized in zeolite topologies. Whereas in ZSM-5 a similar [Cu-O-Cu](2+) active site is located in the intersection of the two 10 membered rings, Cu-MOR provides two distinct local structures, situated in the 8 membered ring windows of the side pockets. Despite their structural similarity, as ascertained by electronic absorption and resonance Raman spectroscopy, the two Cu-O-Cu active sites in Cu-MOR clearly show different kinetic behaviors in selective methane oxidation. This difference in reactivity is too large to be ascribed to subtle differences in the ground states of the Cu-O-Cu sites, indicating the zeolite lattice tunes their reactivity through second-sphere effects. The MOR lattice is therefore functionally analogous to the active site pocket of a metalloenzyme, demonstrating that both the active site and its framework environment contribute to and direct reactivity in transition metal ion-zeolites.

  19. 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

  20. Lapses in skin conductance responding across anatomical sites: Comparison of fingers, feet, forehead, and wrist.

    PubMed

    Payne, Andrew F H; Schell, Anne M; Dawson, Michael E

    2016-07-01

    The fingers are widely accepted as the gold standard for skin conductance (SC) recording, with the feet as a strong alternative. However, there are gaps in the current literature comparing these sites. There is also a great deal of interest in alternative recording sites to permit mobility, but data evaluating these are few and inconsistent. The present report compared multiple sites (fingers, abductor hallucis of the foot, arch of the foot, toes, forehead, and wrist) from 45 college student participants in a short-term sedentary laboratory setting and found large variation in both tonic and phasic SC responses, as well as crucial lapses in responding at nonpalmar sites. Across-site correlations between participants and within participants were also examined. The present data show that, in the laboratory setting employing commonly used recording techniques and stimuli, the nonpalmar sites are generally less responsive than the fingers, and the wrist in particular is the lowest in responding, whereas the toes are most similar to the fingers in responding. Within-participant correlations between the fingers and other sites were greatest for the plantar sites and least for the forehead. PMID:27015847

  1. A Generational Comparison of Social Networking Site Use: The Influence of Age and Social Identity

    ERIC Educational Resources Information Center

    Barker, Valerie

    2012-01-01

    An online survey (N = 256) compared social networking site (SNS) use among younger (millennial: 18-29) and older (baby-boomer: 41-64) subscribers focusing on the influence of collective self-esteem and group identity on motives for SNS use. Younger participants reported higher positive collective self-esteem, social networking site use for peer…

  2. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors.

    PubMed

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M; Kenny, Paul J; Lindstrom, Jon

    2015-05-29

    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.

  3. An investigation of site diversity and comparison with ITU-R recommendations

    NASA Astrophysics Data System (ADS)

    Callaghan, S. A.; Boyes, B.; Couchman, A.; Waight, J.; Walden, C. J.; Ventouras, S.

    2008-08-01

    Earth-space radio systems operating at frequencies of 10 GHz and above are badly attenuated by rain, cloud, and atmospheric gases. As the frequencies of operational systems increase, it becomes increasingly uneconomic to compensate for the effects of fading through the use of a fixed fade margin, hence the implementation of fade mitigation techniques (FMT). The spatial and temporal variation of rain provides the justification for the use of site diversity as an FMT. Site diversity employs two or more ground stations receiving the same satellite signal with a separation distance such that the sites encounter intense rainfall at different times, and switching to the site experiencing the least fading improves system performance considerably. Measurements of the 20.7 GHz beacon carried as part of the Global Broadcast Service (GBS) payload on the U.S. Department of Defense satellite UFO-9 have been made at three sites: two are located in the South of England (˜8 km apart), and the third receiver was located in Scotland. These beacon measurements have produced long term attenuation exceedance and site diversity gain and improvement statistics. This attenuation time series data can simulate the performance of an Earth-space system using site diversity, indicating the optimum method of implementing this FMT. In this paper, unbalanced site diversity is investigated, as this is a more likely scenario than the balanced site diversity modeled by the ITU-R recommendations. This paper also investigates the implementation of site diversity from a commercial context, including cost-benefit analysis and technical feasibility.

  4. 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.

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

    SciTech Connect

    Logan, C.M.; Walden, R.H.; MacFarlane, I.D.

    1995-12-31

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

  7. Sequencing of the amylopullulanase (apu) gene of Thermoanaerobacter ethanolicus 39E, and identification of the active site by site-directed mutagenesis.

    PubMed

    Mathupala, S P; Lowe, S E; Podkovyrov, S M; Zeikus, J G

    1993-08-01

    The complete nucleotide sequence of the gene encoding the dual active amylopullulanase of Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum) was determined. The structural gene (apu) contained a single open reading frame 4443 base pairs in length, corresponding to 1481 amino acids, with an estimated molecular weight of 162,780. Analysis of the deduced sequence of apu with sequences of alpha-amylases and alpha-1,6 debranching enzymes enabled the identification of four conserved regions putatively involved in substrate binding and in catalysis. The conserved regions were localized within a 2.9-kilobase pair gene fragment, which encoded a M(r) 100,000 protein that maintained the dual activities and thermostability of the native enzyme. The catalytic residues of amylopullulanase were tentatively identified by using hydrophobic cluster analysis for comparison of amino acid sequences of amylopullulanase and other amylolytic enzymes. Asp597, Glu626, and Asp703 were individually modified to their respective amide form, or the alternate acid form, and in all cases both alpha-amylase and pullulanase activities were lost, suggesting the possible involvement of 3 residues in a catalytic triad, and the presence of a putative single catalytic site within the enzyme. These findings substantiate amylopullulanase as a new type of amylosaccharidase.

  8. Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

    SciTech Connect

    Farajzadeh, Leila; Hornshøj, Henrik; Momeni, Jamal; Thomsen, Bo; Larsen, Knud; Hedegaard, Jakob; Bendixen, Christian; Madsen, Lone Bruhn

    2013-08-23

    Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i

  9. Unexpected reactivity of 2-fluorolinalyl diphosphate in the active site of crystalline 2-methylisoborneol synthase

    PubMed Central

    Köksal, Mustafa; Chou, Wayne K. W.; Cane, David E.; Christianson, David W.

    2013-01-01

    The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in its unliganded state and in complex with 2 Mg2+ ions and cis-2-fluorogeranyl diphosphate at 1.85 Å and 2.00 Å resolution, respectively. Under normal circumstances, MIBS catalyzes the cyclization of the naturally-occurring, non-canonical 11-carbon isoprenoid substrate, 2-methylgeranyl diphosphate, which first undergoes an ionization-isomerization-ionization sequence through the tertiary diphosphate intermediate 2-methyllinalyl diphosphate to enable subsequent cyclization chemistry. MIBS does not exhibit catalytic activity with 2-fluorogeranyl diphosphate, and we recently reported the crystal structure of MIBS complexed with this unreactive substrate analogue [Köksal, M., Chou, W. K. W., Cane, D. E., Christianson, D. W. (2012) Biochemistry 51, 3011–3020]. However, cocrystallization of MIBS with the fluorinated analogue of the tertiary allylic diphosphate intermediate, 2-fluorolinalyl diphosphate, reveals unexpected reactivity for the intermediate analogue and yields the crystal structure of the complex with the primary allylic diphosphate, 2-fluoroneryl diphosphate. Comparison with the structure of the unliganded enzyme reveals that the crystalline enzyme active site remains partially open, presumably due to the binding of only 2 Mg2+ ions. Assays in solution indicate that MIBS catalyzes the generation of (1R)-(+)-camphor from the substrate 2-fluorolinalyl diphosphate, suggesting that both 2-fluorolinalyl diphosphate and 2-methyllinalyl diphosphate follow the identical cyclization mechanism leading to 2-substituted isoborneol products; however, the initially generated 2-fluoroisoborneol cyclization product is unstable and undergoes elimination of hydrogen fluoride to yield (1R)-(+)-camphor. PMID:23844678

  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. Activity of site-specific endonucleases on complexes of plasmid DNA with multiwalled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Egorova, V. P.; Krylova, H. V.; Lipnevich, I. V.; Veligura, A. A.; Shulitsky, B. G.; Asayonok, A. A.; Vaskovtsev, E. V.

    2016-08-01

    We have synthesized and investigated structural and functional properties of plasmid DNA complexes with multi-walled carbon nanotubes (MWCNTs) for detection of changes in structural state of the plasmid DNA at its recognition by site-specific endonuclease. It has been also established that the site-specific endonuclease is functionally active on the surface of MWCNTs.

  14. 77 FR 5560 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... project proposals on those leases) in identified Wind Energy Areas (WEAs) on the OCS offshore New Jersey... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on the... site assessment plans (SAPs) on those leases. BOEM may issue one or more commercial wind energy...

  15. 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.

  16. SUPPLEMENTARY COMPARISON: COOMET.RI(II)-S1.Rn-222 (169/UA/98): Rn-222 volume activity comparison

    NASA Astrophysics Data System (ADS)

    Skliarov, V.; Röttger, A.; Honig, A.; Korostin, S.; Kuznetsov, S.; Lapenas, A.; Milevsky, V.; Ivaniukovich, A.; Kharitonov, I.; Sepman, S.

    2009-01-01

    According to a first program, a supplementary comparison of Rn-222 volume activity was drawn up as a bilateral supplementary comparison between NSC 'Institute of Metrology', Ukraine, and VNIIFTRI, Russia. It took place in March 2005. In April 2005, at the 5th meeting of COOMET held in Braunschweig (Germany), representatives of these institutes exchanged data which showed the comparability of the national standards of Ukraine and Russia for the check points. During the discussion of the procedure some other institutes decided to join the comparison program, which was extended to BelGIM (Belarus), PTB (Germany), VNIIM (Russia) and RMTC (Latvia). The national standards of volume activity of radon-222 were thus calibrated using one standard radon radiometer as the transfer standard. Results are shown in the Final Report of the comparison. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by COOMET, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

  17. 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

  18. 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.

  19. Comparison of mosquito control programs in seven urban sites in Africa, the Middle East, and the Americas

    PubMed Central

    Impoinvil, Daniel E.; Ahmad, Sajjad; Troyo, Adriana; Keating, Joseph; Githeko, Andrew K.; Mbogo, Charles M; Kibe, Lydiah; Githure, John I.; Gad, Adel M.; Hassan, Ali N.; Orshan, Laor; Warburg, Alon; Calderón-Arguedas, Olger; Sánchez-Loría, Victoria M.; Velit-Suarez, Rosanna; Chadee, Dave D.; Novak, Robert J.; Beier, John C.

    2007-01-01

    Mosquito control programs at seven urban sites in Kenya, Egypt, Israel, Costa Rica, and Trinidad are described and compared. Site-specific urban and disease characteristics, organizational diagrams, and strengths, weaknesses, obstacles and threats (SWOT) analysis tools are used to provide a descriptive assessment of each mosquito control program, and provide a comparison of the factors affecting mosquito abatement. The information for SWOT analysis is collected from surveys, focus group discussions, and personal communication. SWOT analysis identified various issues affecting the efficiency and sustainability of mosquito control operations. The main outcome of our work was the description and comparison of mosquito control operations within the context of each study site’s biological, social, political, management, and economic conditions. The issues identified in this study ranged from lack of inter-sector collaboration to operational issues of mosquito control efforts. A lack of sustainable funding for mosquito control was a common problem for most sites. Many unique problems were also identified, which included lack of mosquito surveillance, lack of law enforcement, and negative consequences of human behavior. Identifying common virtues and shortcomings of mosquito control operations is useful in identifying “best practices” for mosquito control operations, thus leading to better control of mosquito biting and mosquito-borne disease transmission. PMID:17316882

  20. E-ELT seeing and isoplanatic angle: comparison of Aklim site and El Roque de Los Muchachos Observatory

    NASA Astrophysics Data System (ADS)

    Sabil, M.; Benkhaldoun, Z.; Lazrek, M.; Habib, A.; Benhida, A.; Hach, Y.; Elazhari, Y.; Elhalkouj, T.

    2014-07-01

    The new Extremely Large Telescope projects need accurate evaluation of the candidate sites. In this work we present the seeing, free seeing and isoplanatic angle comparison between Aklim site located in Moroccan Anti- Atlas at the geographic coordinates 30°7'39" N, 08 18'39" W, and the Observatorio del Roque de Los Muchachos (ORM), located in La Palma, Canary Islands, at 28°45'00 N, 17°53"10 W, the both sites are pre-selected to house the E-ELT. In this work we present the seeing statistics of (Ɛ), the free seeing (Ɛ free) and the isoplanatic angle ϴ0 measurements at each site, statistics of the mentioned parameters are obtained from the whole data recorded from 09 May 2008 to 09 November 2009 using the Multi Aperture Scintillation Sensor (MASS) - Differential Image Motion Monitor (DIMM) system, compare the common data between the tow sites, more representative results and statistics are shown hereafter.

  1. Comparison of Statistically Modeled Contaminated Soil Volume Estimates and Actual Excavation Volumes at the Maywood FUSRAP Site - 13555

    SciTech Connect

    Moore, James; Hays, David; Quinn, John; Johnson, Robert; Durham, Lisa

    2013-07-01

    As part of the ongoing remediation process at the Maywood Formerly Utilized Sites Remedial Action Program (FUSRAP) properties, Argonne National Laboratory (Argonne) assisted the U.S. Army Corps of Engineers (USACE) New York District by providing contaminated soil volume estimates for the main site area, much of which is fully or partially remediated. As part of the volume estimation process, an initial conceptual site model (ICSM) was prepared for the entire site that captured existing information (with the exception of soil sampling results) pertinent to the possible location of surface and subsurface contamination above cleanup requirements. This ICSM was based on historical anecdotal information, aerial photographs, and the logs from several hundred soil cores that identified the depth of fill material and the depth to bedrock under the site. Specialized geostatistical software developed by Argonne was used to update the ICSM with historical sampling results and down-hole gamma survey information for hundreds of soil core locations. The updating process yielded both a best guess estimate of contamination volumes and a conservative upper bound on the volume estimate that reflected the estimate's uncertainty. Comparison of model results to actual removed soil volumes was conducted on a parcel-by-parcel basis. Where sampling data density was adequate, the actual volume matched the model's average or best guess results. Where contamination was un-characterized and unknown to the model, the actual volume exceeded the model's conservative estimate. Factors affecting volume estimation were identified to assist in planning further excavations. (authors)

  2. 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

  3. Manganese oxide-induced strategy to high-performance iron/nitrogen/carbon electrocatalysts with highly exposed active sites

    NASA Astrophysics Data System (ADS)

    Sun, Tao; Wu, Qiang; Zhuo, Ou; Jiang, Yufei; Bu, Yongfeng; Yang, Lijun; Wang, Xizhang; Hu, Zheng

    2016-04-01

    exposed active sites. Electronic supplementary information (ESI) available: Characterizations on hNCNCs, a series of Fe/N/C catalysts and control catalysts, as well as the performance comparison with those in literatures. See DOI: 10.1039/c6nr00760k

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    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

  6. Comparison of blood volume pulse and skin conductance responses to mental and affective stimuli at different anatomical sites.

    PubMed

    Kushki, Azadeh; Fairley, Jillian; Merja, Satyam; King, Gillian; Chau, Tom

    2011-10-01

    Measurements of blood volume pulse (BVP) and skin conductance are commonly used as indications of psychological arousal in affective computing and human-machine interfaces. To date, palmar surfaces remain the primary site for these measurements. Placement of sensors on palmar surfaces, however, is undesirable when recordings are fraught with motion and pressure artifacts. These artifacts are frequent when the human participant has involuntary movements as in hyperkinetic cerebral palsy. This motivates the use of alternative measurement sites. The present study examined the correlation between measurements of blood volume pulse and skin conductance obtained from three different sites on the body (fingers, toes and ear for BVP; fingers, toes and arch of the foot for skin conductance) in response to cognitive and affective stimuli. The results of this pilot study indicated significant inter-site correlation among signal features derived from different sites, with the exception of BVP amplitude, the number of electrodermal reactions and the slope of the electrodermal activity response. We attribute these differences in part to inter-site discrepancies in local skin conditions, such as skin temperature. Despite these differences, significant changes from baseline were present in the responses to the cognitive and affective stimuli at non-palmar sites, suggesting that these sites may provide viable signal measurements for use in affective computing and human-machine interface applications.

  7. Comparison of blood volume pulse and skin conductance responses to mental and affective stimuli at different anatomical sites

    PubMed Central

    Kushki, Azadeh; Fairley, Jillian; Merja, Satyam; King, Gillian

    2016-01-01

    Measurements of blood volume pulse (BVP) and skin conductance are commonly used as indications of psychological arousal in affective computing and human–machine interfaces. To date, palmar surfaces remain the primary site for these measurements. Placement of sensors on palmar surfaces, however, is undesirable when recordings are fraught with motion and pressure artifacts. These artifacts are frequent when the human participant has involuntary movements as in hyperkinetic cerebral palsy. This motivates the use of alternative measurement sites. The present study examined the correlation between measurements of blood volume pulse and skin conductance obtained from three different sites on the body (fingers, toes and ear for BVP; fingers, toes and arch of the foot for skin conductance) in response to cognitive and affective stimuli. The results of this pilot study indicated significant inter-site correlation among signal features derived from different sites, with the exception of BVP amplitude, the number of electrodermal reactions and the slope of the electrodermal activity response. We attribute these differences in part to inter-site discrepancies in local skin conditions, such as skin temperature. Despite these differences, significant changes from baseline were present in the responses to the cognitive and affective stimuli at non-palmar sites, suggesting that these sites may provide viable signal measurements for use in affective computing and human–machine interface applications. PMID:21849720

  8. Comparison of surface fluxes and conductances at two contrasting sites within the FIFE area

    NASA Technical Reports Server (NTRS)

    Stewart, J. B.; Verma, S. B.

    1992-01-01

    Surface flux measurements from two contrasting sites within the FIFE study area are analyzed. The seasonal variation of midday latent heat fluxes and surface conductances at the two sites was found to be very similar even though the ratio of their average total leaf area indices was greater than two to one. Concurrent measurements made at another six flat sites within the FIFE area confirmed the independence of latent heat flux with respect to leaf area index. The surface conductance stress function related to specific humidity deficit was the same for the two sites, whereas the solar radiation stress function showed a difference of 30 percent at a solar radiation input of 300 W/sq m and the soil moisture stress function showed a difference of 16 percent when half the extractable soil moisture had been taken up. However, when the stress functions were combined to determine the surface conductance, these differences virtually canceled out.

  9. An inter-comparison of PM2.5 at urban and urban background sites: Chemical characterization and source apportionment

    NASA Astrophysics Data System (ADS)

    Cesari, D.; Donateo, A.; Conte, M.; Merico, E.; Giangreco, A.; Giangreco, F.; Contini, D.

    2016-06-01

    A measurement campaign was performed between 04/03/2013 and 17/07/2013 for simultaneous collection of PM2.5 samples in two nearby sites in southeastern Italy: an urban site and an urban background site. PM2.5 at the two sites were similar; however, the chemical composition and the contributions of the main sources were significantly different. The coefficients of divergence (CODs) showed spatial heterogeneity of EC (higher at the urban site because of traffic emissions) and of all metals. Major ions (NH4+, Na+, and SO42 -) and OC had low CODs, suggesting a homogeneous distribution of sea spray, secondary sulfate, and secondary organic matter (SOM = 1.6*OCsec, where OCsec is the secondary OC). The strong correlations between Na+ and Cl-, and the low Cl-/Na+ ratios, suggested the presence of aged sea spray with chloride depletion (about 79% of Cl-) and formation of sodium nitrate at both sites. In both sites, the non-sea-salt sulfate was about 97% of sulfate, and the strong correlation between SO42 - and NH4+ indicated that ammonium was present as ammonium sulfate. However, during advection of Saharan Dust, calcium sulfate was present rather than ammonium sulfate. The source apportionment was performed using the Positive Matrix Factorization comparing outputs of model EPA PMF 3.0 and 5.0 version. Six aerosol sources were identified at both sites: traffic, biomass burning, crustal-resuspended dust, secondary nitrate, marine aerosol, and secondary sulfate. The PMF3.0 model was not completely able, in these sites, to separate marine contribution from secondary nitrate and secondary sulfate from OC, underestimating the marine contribution and overestimating the secondary sulfate with respect to stoichiometric calculations. The application of specific constraints on PMF5.0 provided cleaner profiles, improving the comparison with stoichiometric calculations. The seasonal trends revealed larger biomass burning contributions during the cold period at both sites due to

  10. Probing impact of active site residue mutations on stability and activity of Neisseria polysaccharea amylosucrase.

    PubMed

    Daudé, David; Topham, Christopher M; Remaud-Siméon, Magali; André, Isabelle

    2013-12-01

    The amylosucrase from Neisseria polysaccharea is a transglucosidase from the GH13 family of glycoside-hydrolases that naturally catalyzes the synthesis of α-glucans from the widely available donor sucrose. Interestingly, natural molecular evolution has modeled a dense hydrogen bond network at subsite -1 responsible for the specific recognition of sucrose and conversely, it has loosened interactions at the subsite +1 creating a highly promiscuous subsite +1. The residues forming these subsites are considered to be likely involved in the activity as well as the overall stability of the enzyme. To assess their role, a structure-based approach was followed to reshape the subsite -1. A strategy based on stability change predictions, using the FoldX algorithm, was considered to identify the best candidates for site-directed mutagenesis and guide the construction of a small targeted library. A miniaturized purification protocol was developed and both mutant stability and substrate promiscuity were explored. A range of 8 °C between extreme melting temperature values was observed and some variants were able to synthesize series of oligosaccharides with distributions differing from that of the parental enzyme. The crucial role of subsite -1 was thus highlighted and the biocatalysts generated can now be considered as starting points for further engineering purposes.

  11. Modelling active sites for the Beckmann rearrangement reaction in boron-containing zeolites and their interaction with probe molecules.

    PubMed

    Lezcano-González, Inés; Vidal-Moya, Alejandro; Boronat, Mercedes; Blasco, Teresa; Corma, Avelino

    2010-06-28

    Theoretical calculations and in situ solid state NMR spectroscopy have been combined to get insight on the nature of the active sites for the Beckmann rearrangement reaction in borosilicate zeolites. The interaction of a B site in zeolite Beta with a series of probe molecules (ammonia, pyridine, acetone and water) has been modelled and the (15)N and (11)B NMR isotropic chemical shift of the resulting complexes calculated and compared with experimental in situ NMR results. This approach has allowed validation of the methodology to model the adsorption on a zeolite boron site of molecules of varying basicity which are either protonated or non-protonated. The limitation is that theoretical calculations overestimate the effect of molecular adsorption through hydrogen bonds on the calculated isotropic (11)B NMR chemical shift.Theoretical and experimental results on the adsorption of acetophenone and cyclohexanone oximes on zeolite B-Beta indicate that Brønsted acid sites protonate the oximes, changing the boron coordination from trigonal to tetrahedral. Comparison of theoretical and experimental (15)N NMR chemical shifts of the adsorbed amides (acetanilide and epsilon-caprolactam) indicates that they are non-protonated, and the (11)B NMR spectra show that, as expected, boron remains in trigonal coordination with an isotropic delta(11)B(exp) which differs from the calculated value delta(11)B(calc).

  12. Modelling active sites for the Beckmann rearrangement reaction in boron-containing zeolites and their interaction with probe molecules.

    PubMed

    Lezcano-González, Inés; Vidal-Moya, Alejandro; Boronat, Mercedes; Blasco, Teresa; Corma, Avelino

    2010-06-28

    Theoretical calculations and in situ solid state NMR spectroscopy have been combined to get insight on the nature of the active sites for the Beckmann rearrangement reaction in borosilicate zeolites. The interaction of a B site in zeolite Beta with a series of probe molecules (ammonia, pyridine, acetone and water) has been modelled and the (15)N and (11)B NMR isotropic chemical shift of the resulting complexes calculated and compared with experimental in situ NMR results. This approach has allowed validation of the methodology to model the adsorption on a zeolite boron site of molecules of varying basicity which are either protonated or non-protonated. The limitation is that theoretical calculations overestimate the effect of molecular adsorption through hydrogen bonds on the calculated isotropic (11)B NMR chemical shift.Theoretical and experimental results on the adsorption of acetophenone and cyclohexanone oximes on zeolite B-Beta indicate that Brønsted acid sites protonate the oximes, changing the boron coordination from trigonal to tetrahedral. Comparison of theoretical and experimental (15)N NMR chemical shifts of the adsorbed amides (acetanilide and epsilon-caprolactam) indicates that they are non-protonated, and the (11)B NMR spectra show that, as expected, boron remains in trigonal coordination with an isotropic delta(11)B(exp) which differs from the calculated value delta(11)B(calc). PMID:20454729

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

    PubMed

    Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

    2014-04-01

    The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins.

  14. 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.

  15. Nuclear waste: Status of DOE`s nuclear waste site characterization activities

    SciTech Connect

    1987-12-31

    Three potential nuclear waste repository sites have been selected to carry out characterization activities-the detailed geological testing to determine the suitability of each site as a repository. The sites are Hanford in south-central Washington State, Yucca Mountain in southern Nevada, and Deaf Smith in the Texas Panhandle. Two key issues affecting the total program are the estimations of the site characterization completion data and costs and DOE`s relationship with the Nuclear Regulatory Commission which has been limited and its relations with affected states and Indian tribes which continue to be difficult.

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

    NASA Astrophysics Data System (ADS)

    Miyamoto, Daisuke; Ichikuni, Nobuyuki; Shimazu, Shogo

    2007-02-01

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

  17. VS30, site amplifications and some comparisons: The Adapazari (Turkey) case

    NASA Astrophysics Data System (ADS)

    Ozcep, Tazegul; Ozcep, Ferhat; Ozel, Oguz

    The aim of this study was to investigate the role of VS30 in site amplifications in the Adapazari region, Turkey. To fulfil this aim, amplifications from VS30 measurements were compared with earthquake data for different soil types in the seismic design codes. The Adapazari area was selected as the study area, and shear-wave velocity distribution was obtained by the multichannel analysis of surface waves (MASWs) method at 100 sites for the top 50 m of soil. Aftershock data following the Mw 7.4 Izmit earthquake of 17 August 1999 gave magnitudes between 4.0 and 5.6 at six stations installed in and around the Adapazari Basin, at Babalı, Şeker, Genç, Hastane, Toyota and Imar. This data was used to estimate site amplifications by the reference-station method. In addition, the fundamental periods of the station sites were estimated by the single station method. Site classifications based on VS30 in the seismic design codes were compared with the fundamental periods and amplification values. It was found that site amplifications (from earthquake data) and relevant spectra (from VS30) are not in good agreement for soils in Adapazari (Turkey).

  18. Quantitative comparison of cis-regulatory element (CRE) activities in transgenic Drosophila melanogaster.

    PubMed

    Rogers, William A; Williams, Thomas M

    2011-01-01

    Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism's program for development, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE's activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites. This capability has made the quantitative measurement of gene and, relevant here, CRE activity feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at

  19. 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

  20. Mechanistic and bioinformatic investigation of a conserved active site helix in α-isopropylmalate synthase from Mycobacterium tuberculosis, a member of the DRE-TIM metallolyase superfamily.

    PubMed

    Casey, Ashley K; Hicks, Michael A; Johnson, Jordyn L; Babbitt, Patricia C; Frantom, Patrick A

    2014-05-13

    The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily.

  1. Transcriptional activation through ETS domain binding sites in the cytochrome c oxidase subunit IV gene

    SciTech Connect

    Virbasius, J.V.; Scarpulla, R.C. )

    1991-11-01

    A mutational analysis of the rat cytochrome c oxidase subunit IV (RCO4) promoter region revealed the presence of a major control element consisting of a tandemly repeated pair of binding sites for a nuclear factor from HeLa cells. This factor was designated NRF-2 (nuclear respiratory factor 2) because a functional recognition site was also found in the human ATP synthase {beta}-subunit gene. Deletion or site-directed point mutations of the NRF-2 binding sites in the RCO4 promoter resulted in substantial loss of transcriptional activity, and synthetic oligomers of the NRF-2 binding sites from both genes stimulated a heterologous promoter when cloned in cis. NRF-2 binding a transcriptional activation required a purine-rich core sequence, GGAA. This motif is characteristic of the recognition site for a family of activators referred to as ETS domain proteins because of the similarity within their DNA-binding domains to the ets-1 proto-oncogene product. NRF-2 recognized an authentic Ets-1 site within the Moloney murine sarcoma virus long terminal repeat, and this site was able to compete for NRF-2 binding to the RCO4 promoter sequence. However, in contrast to Ets-1, which appears to be exclusive to lymphoid tissues, NRF-2 has the broad tissue distribution expected of a regulator of respiratory chain expression.

  2. Comparison of surface fluxes and boundary-layer measurements at Arctic terrestrial sites

    NASA Astrophysics Data System (ADS)

    Grachev, Andrey; Uttal, Taneil; Persson, Ola; Stone, Robert; Crepinsek, Sara; Albee, Robert; Makshtas, Alexander; Kustov, Vasily; Repina, Irina; Artamonov, Arseniy

    2014-05-01

    Observational evidence suggests that atmospheric energy fluxes are a major contributor to the decrease of the Arctic pack ice, seasonal land snow cover and the warming of the surrounding land areas and permafrost layers. To better understand the atmosphere-surface exchange mechanisms, improve models, and to diagnose climate variability in the Arctic, accurate measurements are required of all components of the net surface energy budget and the carbon dioxide cycle over representative areas and over multiple years. This study analyzes and discusses variability of surface fluxes and basic meteorological parameters based on measurements made at several long-term research observatories near the coast of the Arctic Ocean located in USA (Barrow), Canada (Eureka), and Russia (Tiksi). Tower-based eddy covariance and solar radiation measurements provide a long-term near continuous temporal record of hourly average mass and energy fluxes respectively. The turbulent fluxes of the momentum, sensible heat, water vapor, and carbon dioxide are supported by additional atmospheric and surface/snow/permafrost measurements (mean wind speed, air temperature and humidity, upwelling and downwelling short-wave and long-wave atmospheric and surface radiation, snow depth, surface albedo, soil heat flux, active layer temperature profiles etc.) In this study we compare annual cycles of surface fluxes including solar radiation and other ancillary data to describe four seasons in the Arctic including spring onset of melt and fall onset of snow accumulation. Particular interest is a transition through freezing point, i.e. during transition from winter to spring and from summer to fall, when the carbon dioxide and/or water vapor turbulent fluxes change their direction. According to our data, in a summer period observed temporal variability of the carbon dioxide flux was generally in anti-phase with water vapor flux (downward CO2 flux and upward H2O flux). On average the turbulent flux of carbon

  3. Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase.

    PubMed

    Fafarman, Aaron T; Sigala, Paul A; Schwans, Jason P; Fenn, Timothy D; Herschlag, Daniel; Boxer, Steven G

    2012-02-01

    Understanding the electrostatic forces and features within highly heterogeneous, anisotropic, and chemically complex enzyme active sites and their connection to biological catalysis remains a longstanding challenge, in part due to the paucity of incisive experimental probes of electrostatic properties within proteins. To quantitatively assess the landscape of electrostatic fields at discrete locations and orientations within an enzyme active site, we have incorporated site-specific thiocyanate vibrational probes into multiple positions within bacterial ketosteroid isomerase. A battery of X-ray crystallographic, vibrational Stark spectroscopy, and NMR studies revealed electrostatic field heterogeneity of 8 MV/cm between active site probe locations and widely differing sensitivities of discrete probes to common electrostatic perturbations from mutation, ligand binding, and pH changes. Electrostatic calculations based on active site ionization states assigned by literature precedent and computational pK(a) prediction were unable to quantitatively account for the observed vibrational band shifts. However, electrostatic models of the D40N mutant gave qualitative agreement with the observed vibrational effects when an unusual ionization of an active site tyrosine with a pK(a) near 7 was included. UV-absorbance and (13)C NMR experiments confirmed the presence of a tyrosinate in the active site, in agreement with electrostatic models. This work provides the most direct measure of the heterogeneous and anisotropic nature of the electrostatic environment within an enzyme active site, and these measurements provide incisive benchmarks for further developing accurate computational models and a foundation for future tests of electrostatics in enzymatic catalysis.

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

    PubMed

    Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

    2015-04-21

    This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP

  5. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity.

    PubMed

    Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H

    2016-09-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes.

  6. 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.

  7. An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein.

    PubMed

    Hirschi, Alexander; Cecchini, Matthew; Steinhardt, Rachel C; Schamber, Michael R; Dick, Frederick A; Rubin, Seth M

    2010-09-01

    The phosphorylation state and corresponding activity of the retinoblastoma tumor suppressor protein (Rb) are modulated by a balance of kinase and phosphatase activities. Here we characterize the association of Rb with the catalytic subunit of protein phosphatase 1 (PP1c). A crystal structure identifies an enzyme docking site in the Rb C-terminal domain that is required for efficient PP1c activity toward Rb. The phosphatase docking site overlaps with the known docking site for cyclin-dependent kinase (Cdk), and PP1 competition with Cdk-cyclins for Rb binding is sufficient to retain Rb activity and block cell-cycle advancement. These results provide the first detailed molecular insights into Rb activation and establish a novel mechanism for Rb regulation in which kinase and phosphatase compete for substrate docking. PMID:20694007

  8. Active site nanospace of aminoacyl tRNA synthetase: difference between the class I and class II synthetases.

    PubMed

    Dutta, Saheb; Choudhury, Kaberi; Banik, Sindrila Dutta; Nandi, Nilashis

    2014-03-01

    The present work is aimed at understanding the origin of the difference in the molecular organization of the active site nanospaces of the class I and class II aminoacyl tRNA synthetases (aaRSs) which are tunnel-like structures. The active site encloses the cognate amino acid (AA) and the adenosine triphosphate (ATP) to carry out aminoacylation reaction. Comparison of the structures of the active site of the class I and class II (aaRSs) shows that the nanodimensional tunnels are curved in opposite directions in the two classes. We investigated the origin of this difference using quantum mechanical computation of electrostatic potential (ESP) of substrates, surrounding residues and ions, using Atoms in Molecule (AIM) Theory and charge population analysis. We show that the difference is principally due to the variation in the spatial charge distribution of ATP in the two classes which correspond to extended and bent conformations of ATP. The present computation shows that the most feasible pathway for nucleophilic attack to alphaP is oppositely directed for class I and class II aaRSs. The available crystal structures show that the cognate AA is indeed located along the channel favorable for nucleophilic attack as predicted by the ESP analysis. It is also shown that the direction of the channel changes its orientation when the orientation of ATP is changed from extended to a bent like structure. We further used the AIM theory to confirm the direction of the approach of AA in each case and the results corroborate the results from the ESP analysis. The opposite curvatures of the active site nanospaces in class I and class II aaRSs are related with the influence of the charge distributions of the extended and bent conformations of ATP, respectively. The results of the computation of electrostatic potential by successive addition of active site residues show that their roles on the reaction are similar in both classes despite the difference in the organization of the

  9. 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.

  10. An inter-comparison of PM10 source apportionment using PCA and PMF receptor models in three European sites.

    PubMed

    Cesari, Daniela; Amato, F; Pandolfi, M; Alastuey, A; Querol, X; Contini, D

    2016-08-01

    Source apportionment of aerosol is an important approach to investigate aerosol formation and transformation processes as well as to assess appropriate mitigation strategies and to investigate causes of non-compliance with air quality standards (Directive 2008/50/CE). Receptor models (RMs) based on chemical composition of aerosol measured at specific sites are a useful, and widely used, tool to perform source apportionment. However, an analysis of available studies in the scientific literature reveals heterogeneities in the approaches used, in terms of "working variables" such as the number of samples in the dataset and the number of chemical species used as well as in the modeling tools used. In this work, an inter-comparison of PM10 source apportionment results obtained at three European measurement sites is presented, using two receptor models: principal component analysis coupled with multi-linear regression analysis (PCA-MLRA) and positive matrix factorization (PMF). The inter-comparison focuses on source identification, quantification of source contribution to PM10, robustness of the results, and how these are influenced by the number of chemical species available in the datasets. Results show very similar component/factor profiles identified by PCA and PMF, with some discrepancies in the number of factors. The PMF model appears to be more suitable to separate secondary sulfate and secondary nitrate with respect to PCA at least in the datasets analyzed. Further, some difficulties have been observed with PCA in separating industrial and heavy oil combustion contributions. Commonly at all sites, the crustal contributions found with PCA were larger than those found with PMF, and the secondary inorganic aerosol contributions found by PCA were lower than those found by PMF. Site-dependent differences were also observed for traffic and marine contributions. The inter-comparison of source apportionment performed on complete datasets (using the full range of

  11. An inter-comparison of PM10 source apportionment using PCA and PMF receptor models in three European sites.

    PubMed

    Cesari, Daniela; Amato, F; Pandolfi, M; Alastuey, A; Querol, X; Contini, D

    2016-08-01

    Source apportionment of aerosol is an important approach to investigate aerosol formation and transformation processes as well as to assess appropriate mitigation strategies and to investigate causes of non-compliance with air quality standards (Directive 2008/50/CE). Receptor models (RMs) based on chemical composition of aerosol measured at specific sites are a useful, and widely used, tool to perform source apportionment. However, an analysis of available studies in the scientific literature reveals heterogeneities in the approaches used, in terms of "working variables" such as the number of samples in the dataset and the number of chemical species used as well as in the modeling tools used. In this work, an inter-comparison of PM10 source apportionment results obtained at three European measurement sites is presented, using two receptor models: principal component analysis coupled with multi-linear regression analysis (PCA-MLRA) and positive matrix factorization (PMF). The inter-comparison focuses on source identification, quantification of source contribution to PM10, robustness of the results, and how these are influenced by the number of chemical species available in the datasets. Results show very similar component/factor profiles identified by PCA and PMF, with some discrepancies in the number of factors. The PMF model appears to be more suitable to separate secondary sulfate and secondary nitrate with respect to PCA at least in the datasets analyzed. Further, some difficulties have been observed with PCA in separating industrial and heavy oil combustion contributions. Commonly at all sites, the crustal contributions found with PCA were larger than those found with PMF, and the secondary inorganic aerosol contributions found by PCA were lower than those found by PMF. Site-dependent differences were also observed for traffic and marine contributions. The inter-comparison of source apportionment performed on complete datasets (using the full range of

  12. On splice site prediction using weight array models: a comparison of smoothing techniques

    NASA Astrophysics Data System (ADS)

    Taher, Leila; Meinicke, Peter; Morgenstern, Burkhard

    2007-11-01

    In most eukaryotic genes, protein-coding exons are separated by non-coding introns which are removed from the primary transcript by a process called "splicing". The positions where introns are cut and exons are spliced together are called "splice sites". Thus, computational prediction of splice sites is crucial for gene finding in eukaryotes. Weight array models are a powerful probabilistic approach to splice site detection. Parameters for these models are usually derived from m-tuple frequencies in trusted training data and subsequently smoothed to avoid zero probabilities. In this study we compare three different ways of parameter estimation for m-tuple frequencies, namely (a) non-smoothed probability estimation, (b) standard pseudo counts and (c) a Gaussian smoothing procedure that we recently developed.

  13. Summary and Comparison of Multiphase Streambed Scour Analysis at Selected Bridge Sites in Alaska

    USGS Publications Warehouse

    Conaway, Jeffrey S.

    2004-01-01

    The U.S. Geological Survey and the Alaska Department of Transportation and Public Facilities undertook a cooperative multiphase study of streambed scour at selected bridges in Alaska beginning in 1994. Of the 325 bridges analyzed for susceptibility to scour in the preliminary phase, 54 bridges were selected for a more intensive analysis that included site investigations. Cross-section geometry and hydraulic properties for each site in this study were determined from field surveys and bridge plans. Water-surface profiles were calculated for the 100- and 500-year floods using the Hydrologic Engineering Center?s River Analysis System and scour depths were calculated using methods recommended by the Federal Highway Administration. Computed contraction-scour depths for the 100- and 500-year recurrence-interval discharges exceeded 5 feet at six bridges, and pier-scour depths exceeded 10 feet at 24 bridges. Complex pier-scour computations were made at 10 locations where the computed contraction-scour depths would expose pier footings. Pressure scour was evaluated at three bridges where the modeled flood water-surface elevations intersected the bridge structure. Site investigation at the 54 scour-critical bridges was used to evaluate the effectiveness of the preliminary scour analysis. Values for channel-flow angle of attack and approach-channel width were estimated from bridge survey plans for the preliminary study and were measured during a site investigation for this study. These two variables account for changes in scour depths between the preliminary analysis and subsequent reanalysis for most sites. Site investigation is needed for best estimates of scour at bridges with survey plans that indicate a channel-flow angle of attack and for locations where survey plans did not include sufficient channel geometry upstream of the bridge.

  14. 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

  15. A Comparison of the Soil Bioload of Mars Analog Sites Using a Novel Life Detection Instrument.

    NASA Astrophysics Data System (ADS)

    Smith, H.; Powers, L. S.; McKay, C. P.; Lloyd, C.; Duncan, A.; Rich, S.

    2007-12-01

    In this presentation we compare the microbial bioload at Mars Analog Sites, such as the Moab, Mojave, and Atacama Deserts as well as high altitude volcanic environments. A novel technique using intrinsic fluorescence for life detection was implemented in-situ to quantify total soil biomass. Soil samples were collected to compare in-situ techniques with traditional microbial assays, such as culturing. The Mars Analog sites differ in soil bioload, but show similarities in microbial patchiness distribution. In addition to the microbial work we also describe the novel instrument used to detect and quantify the total soil bioload.

  16. 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

  17. 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

  18. 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

  19. Perceptions of Web Site Design Characteristics: A Malaysian/Australian Comparison.

    ERIC Educational Resources Information Center

    Fink, Dieter; Laupase, Ricky

    2000-01-01

    Compares the perceptions of Malaysians and Australians for four Web site design characteristics--atmospherics, news stories, signs, and products and services--as part of the integrated Internet marketing model. Hypothesizes that the predominant culture is not generalized to another culture, discusses validity and reliability, and suggest further…

  20. Web Sites Face Hurdles in Promising Students Unbiased Loan-Rate Comparisons

    ERIC Educational Resources Information Center

    Basken, Paul

    2007-01-01

    Someday students seeking a college loan might go to a Web site, fill out a brief questionnaire, and get connected to the lender with the best possible rate. Such a system would bring cheer to students seeking straight answers, lenders seeking fair competition, and colleges seeking relief from investigations led by Attorney General Andrew M. Cuomo…

  1. Comparisons of CAP88PC version 2.0 default parameters to site specific inputs

    SciTech Connect

    Lehto, M. A.; Courtney, J. C.; Charter, N.; Egan, T.

    2000-03-02

    The effects of varying the input for the CAP88PC Version 2.0 program on the total effective dose equivalents (TEDEs) were determined for hypothetical releases from the Hot Fuel Examination Facility (HFEF) located at the Argonne National Laboratory site on the Idaho National Engineering and Environmental Laboratory (INEEL). Values for site specific meteorological conditions and agricultural production parameters were determined for the 80 km radius surrounding the HFEF. Four nuclides, {sup 3}H, {sup 85}Kr, {sup 129}I, and {sup 137}Cs (with its short lived progeny, {sup 137m}Ba) were selected for this study; these are the radioactive materials most likely to be released from HFEF under normal or abnormal operating conditions. Use of site specific meteorological parameters of annual precipitation, average temperature, and the height of the inversion layer decreased the TEDE from {sup 137}Cs-{sup 137m}Ba up to 36%; reductions for other nuclides were less than 3%. Use of the site specific agricultural parameters reduced TEDE values between 7% and 49%, depending on the nuclide. Reductions are associated with decreased committed effective dose equivalents (CEDEs) from the ingestion pathway. This is not surprising since the HFEF is located well within the INEEL exclusion area, and the surrounding area closest to the release point is a high desert with limited agricultural diversity. Livestock and milk production are important in some counties at distances greater than 30 km from the HFEF.

  2. Faculty supervision of NP program practicums: A comparison of rural and urban site differences.

    PubMed

    Kaplan, Louise; Klein, Tracy; Skillman, Susan; Andrilla, C Holly

    2016-06-16

    Results of a survey of NP education programs were analyzed to describe factors influencing clinical placement of students in rural clinical rotations, methods used to evaluate NP students in their clinical rotations, and whether the frequency and type of faculty evaluations differed by urban or rural clinical sites. PMID:27327427

  3. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... (20″×14″) upright format signs specified in 29 CFR 1910.145(d)(4) and this paragraph; and (iii... 40 Protection of Environment 8 2011-07-01 2011-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an...

  4. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (20″×14″) upright format signs specified in 29 CFR 1910.145(d)(4) and this paragraph; and (iii... 40 Protection of Environment 8 2010-07-01 2010-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an...

  5. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... (20″×14″) upright format signs specified in 29 CFR 1910.145(d)(4) and this paragraph; and (iii... 40 Protection of Environment 9 2013-07-01 2013-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an...

  6. 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... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on the... Activities on the Atlantic OCS Offshore RI and MA'' to: Program Manager, Office of Renewable Energy...

  7. 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.

  8. Nitrogen dynamics in peat bogs: Comparison of sites with contrasting pollution levels (Central Europe)

    NASA Astrophysics Data System (ADS)

    Novak, Martin; Bohdalkova, Leona; Stepanova, Marketa; Vile, Melanie A.; Wieder, Kelman R.

    2013-04-01

    Nitrogen belongs to chemical elements whose biogeochemical cycles are most heavily disturbed by human activities, and large regions worlwide experience elevated depositions of reactive N (NO3-, NH4+). Peatlands contain as much as 15 % of the world's soil N. It it is unclear whether fertilizing by anthopogenic N will lead to higher storage of C in wetlands. Elevated N input may lead to both higher net primary productivity, but will also augment microbial decomposition. Here we discuss two aspects of N cycling in Sphagnum-dominated bogs in the Czech Republic, an area characterized by a steep north-south pollution gradient and high annual N deposition (60 kg ha-1). These two aspects are N inventory in 210Pb-dated peat cores, and post-depositional mobility of N in peat. We compared the N inventory in two Czech bogs, differing in pollution, with cumulative atmospheric N input. We hypothesized that the total amount of N in the peat cores would be smaller than the cumulative N input (leaching of excess N from the bog, denitrification). The two bogs were VJ (industrial north) and CB (rural south). The investigated period was 1885-2002. The total amount of N was 4020 kg ha-1 at VJ and 1530 kg ha-1 at CB. Peat in the north contained 2.6 times more N than in the south. Historical rates of N deposition in the Czech Republic are well known (numerous papers by Kopacek). To estimate cumulative N inputs into the bogs, we also used the monthly N depositions between 1994 and 2002, measured in two nearby catchments. The estimated cumulative atmospheric N input was 1350 kg ha-1 at VJ, and 530 kg ha-1 at CB. In both cases, the amount of N found in peat was 3 times higher than the estimated atmospheric N input. Such high storage of N in peat is surprising. Post-depositional mobility of N may help to explain the discrepancies between atmospheric N inputs and N storage in peat. We found two-fold evidence for post-depositional mobility of N. Maximum N concentrations at VJ were observed in

  9. Comparison of Forest Soil Carbon Dynamics at Five Sites Along a Latitudinal Gradient

    SciTech Connect

    Garten Jr, Charles T

    2011-01-01

    Carbon stocks, and C:N ratios, were measured in the forest floor, mineral soil, and two mineral soil fractions (particulate and mineral-associated organic matter, POM and MOM, respectively) at five forest sites, ranging from 60 to 100 years old, along a latitudinal gradient in the eastern United States. Sampling at four sites was replicated over two consecutive years. For many measurements (like forest floor carbon stocks, cumulative soil organic carbon stocks to 20 cm, and the fraction of whole soil carbon in POM), there was no significant difference between years at each site despite the use of somewhat different sampling methods. With one exception, forest floor and mineral soil carbon stocks increased from warm, southern, sites (with fine-textured soils) to northern, cool, sites (with more coarse-textured soils). The exception was a northern site, with less than 10% silt-clay content, that had a soil organic carbon stock similar to those measured at southern sites. Soil carbon at each site was partitioned into two pools (labile and stable) on the basis of carbon measured in the forest floor and POM and MOM fractions from the mineral soil. A two-compartment steady-state model, with randomly varying parameter values, was used in probabilistic calculations to estimate the turnover time of labile soil organic carbon (MRTU) and the annual transfer of labile carbon to stable carbon (k2) at each site in two different years. Based on empirical data, the turnover time of stable soil carbon (MRTS) was determined by mean annual temperature and increased from 30 to 100 years from south to north. Moving from south to north, MRTU increased from approximately 5 to 14 years. Consistent with prior studies, 13C enrichment factors ( ) from the Rayleigh equation, that describe the rate of change in 13C through the soil profile, were an indicator of soil carbon turnover times along the latitudinal gradient. Consistent with its role in stabilization of soil organic carbon, silt

  10. 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.

  11. 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

  12. School Counseling Intern Roles: Exploration of Activities and Comparison to the ASCA National Model

    ERIC Educational Resources Information Center

    Leuwerke, Wade C.; Bruinekool, R. Matthew; Lane, Amy

    2008-01-01

    Examination of 6,556 hours of school counselor interns' activity logs provided a detailed description of roles and activities. Comparison of counselor intern activities to the ASCA (2005) National Model found consistency between responsive services at the elementary level and both responsive services and guidance curriculum at the middle school…

  13. Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge.

    PubMed

    Tara, S; Elcock, A H; Kirchhoff, P D; Briggs, J M; Radic, Z; Taylor, P; McCammon, J A

    1998-12-01

    It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant kon, for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction.

  14. Advanced Imaging for Glaucoma Study: Design, Baseline Characteristics, and Inter-Site Comparison

    PubMed Central

    Le, Phuc V.; Zhang, Xinbo; Francis, Brian A.; Varma, Rohit; Greenfield, David S.; Schuman, Joel S.; Loewen, Nils; Huang, David

    2014-01-01

    Purpose To report the baseline characteristics of the participants in the Advanced Imaging for Glaucoma Study. To compare the participating sites for variations among subjects and the performance of imaging instruments. Design Multi-center longitudinal observational cohort study Methods A total of 788 participants (1,329 eyes) were enrolled from three academic referral centers. There were 145 participants (289 eyes) in the normal group, 394 participants (663 eyes) in the glaucoma suspect/preperimetric glaucoma group, and 249 participants (377 eyes) in the perimetric glaucoma group. Participants underwent a full clinical exam, standard automated perimetry, and imaging with time-domain and Fourier-domain optical coherence tomography (OCT), scanning laser polarimetry, and confocal scanning laser ophthalmoscopy. Main Outcome Measures The baseline average, population standard deviation, and repeatability of imaging-derived anatomic variables were reported for each technology and center. Results Compared to the normal participants, glaucoma suspect/preperimetric glaucoma and perimetric glaucoma groups had significantly reduced anatomic measurements. Repeatability of nerve fiber layer thickness was best for Fourier-domain OCT (overall coefficient of variation < 2%), followed by time-domain OCT (coefficient of variation 2-2.9%), scanning laser polarimetry (coefficient of variation 2.6-4.5%), and confocal scanning laser ophthalmoscopy rim area (coefficient of variation 4.2-7.6%). A mixed-effects model showed that the differences between sites was less than 25 percent of the variation within groups and less than the differences between the normal and glaucoma suspect/preperimetric glaucoma group. Conclusions Site-to-site variation was smaller than both the variation within groups and the changes due to glaucoma. Therefore pooling of participants between sites is appropriate. PMID:25447111

  15. High-throughput functional comparison of promoter and enhancer activities.

    PubMed

    Nguyen, Thomas A; Jones, Richard D; Snavely, Andrew R; Pfenning, Andreas R; Kirchner, Rory; Hemberg, Martin; Gray, Jesse M

    2016-08-01

    Promoters initiate RNA synthesis, and enhancers stimulate promoter activity. Whether promoter and enhancer activities are encoded distinctly in DNA sequences is unknown. We measured the enhancer and promoter activities of thousands of DNA fragments transduced into mouse neurons. We focused on genomic loci bound by the neuronal activity-regulated coactivator CREBBP, and we measured enhancer and promoter activities both before and after neuronal activation. We find that the same sequences typically encode both enhancer and promoter activities. However, gene promoters generate more promoter activity than distal enhancers, despite generating similar enhancer activity. Surprisingly, the greater promoter activity of gene promoters is not due to conventional core promoter elements or splicing signals. Instead, we find that particular transcription factor binding motifs are intrinsically biased toward the generation of promoter activity, whereas others are not. Although the specific biases we observe may be dependent on experimental or cellular context, our results suggest that gene promoters are distinguished from distal enhancers by specific complements of transcriptional activators. PMID:27311442

  16. 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.

  17. 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

  18. 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

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

    PubMed

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

    2015-12-16

    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.

  20. Dual evaluation of some novel 2-amino-substituted coumarinylthiazoles as anti-inflammatory-antimicrobial agents and their docking studies with COX-1/COX-2 active sites.

    PubMed

    Chandak, Navneet; Kumar, Pawan; Kaushik, Pawan; Varshney, Parul; Sharma, Chetan; Kaushik, Dhirender; Jain, Sudha; Aneja, Kamal R; Sharma, Pawan K

    2014-08-01

    Synthesis of total eighteen 2-amino-substituted 4-coumarinylthiazoles including sixteen new compounds (3a-o and 5b) bearing the benzenesulfonamide moiety is described in the present report. All the synthesized target compounds were examined for their in vivo anti-inflammatory (AI) activity and in vitro antimicrobial activity. Results revealed that six compounds (3 d, 3 f, 3 g, 3 h, 3 j and 3 n) exhibited pronounced anti-inflammatory activity comparable to the standard drug indomethacin. AI results were further confirmed by the docking studies of the most active (3n) and the least active compound (3a) with COX-1 and COX-2 active sites. In addition, most of the compounds exhibited moderate antimicrobial activity against Gram-positive bacteria as well as fungal yeast, S. cervisiae. Comparison between 3 and 5 indicated that incorporation of additional substituted pyrazole nucleus into the scaffold significantly enhanced AI activity. PMID:23777557

  1. Comparison of N2O Emissions from Soils at Three Temperate Agricultural Sites

    NASA Technical Reports Server (NTRS)

    Frolking, S. E.; Moiser, A. R.; Ojima, D. S.; Li, C.; Parton, W. J.; Potter, C. S.; Priesack, E.; Stenger, R.; Haberbosch, C.; Dorsch, P.; Peterson, David L. (Technical Monitor)

    1997-01-01

    Nitrous oxide (N2O) flux simulations by four models were compared with year-round field measurements from five temperate agricultural sites in three countries. The field sites included an unfertilized, semi-arid rangeland with low N2O fluxes in eastern Colorado, USA; two fertilizer treatments (urea and nitrate) on a fertilized grass ley cut for silage in Scotland; and two fertilized, cultivated crop fields in Germany where N2O loss during the winter was quite high. The models used were daily trace gas versions of the CENTURY model, DNDC, ExpertN, and the NASA-Ames version of the CASA model. These models included similar components (soil physics, decomposition, plant growth, and nitrogen transformations), but in some cases used very different algorithms for these processes. All models generated similar results for the general cycling of nitrogen through the agro-ecosystems, but simulated nitrogen trace gas fluxes were quite different. In most cases the simulated N20 fluxes were within a factor of about 2 of the observed annual fluxes, but even when models produced similar N2O fluxes they often produced very different estimates of gaseous N loss as nitric oxide (NO), dinitrogen (N2), and ammonia (NH3). Accurate simulation of soil moisture appears to be a key requirement for reliable simulation of N2O emissions. All models simulated the general pattern of low background fluxes with high fluxes following fertilization at the Scottish sites, but they could not (or were not designed to) accurately capture the observed effects of different fertilizer types on N2O flux. None of the models were able to reliably generate large pulses of N2O during brief winter thaws that were observed at the two German sites. All models except DNDC simulated very low N2O fluxes for the dry site in Colorado. The US Trace Gas Network (TRAGNET) has provided a mechanism for this model and site intercomparison. Additional intercomparisons are needed with these and other models and additional data

  2. Biogeochemical Process Comparison of the Five USGS Water, Energy, and Biogeochemical Budget (WEBB) Sites

    NASA Astrophysics Data System (ADS)

    Shanley, J. B.; Peters, N. E.; Aulenbach, B. T.; Blum, A. E.; Campbell, D. H.; Clow, D. W.; Larsen, M. C.; Mast, M. A.; Stallard, R. F.; Troester, J. W.; Walker, J. F.; Webb, R. M.; White, A. F.

    2001-12-01

    Input - output budgets (in wet deposition and streamwater) have been constructed for water and major solutes at the five USGS Water, Energy, and Biogeochemical Budget (WEBB) sites for the period 1992-97 (Peters et al., 2000). In this poster we interpret the net chemical fluxes to identify the controlling biogeochemical processes, as influenced by the strong physical and biological contrasts (climate, geology, physiography, and vegetation types) in the five diverse environments. The five sites are: Allequash Creek, Wisconsin (low-relief humid continental forest); Andrews Creek, Colorado (cold alpine, taiga/tundra, and subalpine boreal forest); Icacos River, Puerto Rico (lower montane, wet tropical forest); Panola Mountain, Georgia (humid subtropical piedmont forest); and Sleepers River, Vermont (humid northern hardwood forest). Base cations and Si produced by chemical weathering displayed a net export at each site. The magnitude and stoichiometry of export reflects mineralogy, climate (temperature and rainfall), and water residence time in the subsurface. The lowest and highest mass export generally was for Andrews Creek and Icacos River, respectively, consistent with their extreme mean annual temperatures (0/degC in Colorado to 21/degC in Puerto Rico) and the limited residence time of meltwater at Andrews Creek. Calcite in bedrock at the three coldest watersheds caused somewhat higher relative export of Ca, especially at Sleepers River where calcite weathering is a dominant control on stream chemistry. In contrast, the high Mg content of the volcaniclastic rocks at Icacos River and glacial deposits at Allequash Creek caused disproportionately high Mg export relative to the other sites. Relatively high Na export at Panola Mountain and K export at Sleepers River are probably caused by plagioclase and biotite weathering, respectively. SO4 is retained at the two warmest sites, Panola Mountain and Icacos River. SO4 adsorption is known to limit SO4- export in highly

  3. RADIOLYTIC HYDROGEN GENERATION INSAVANNAH RIVER SITE (SRS) HIGH LEVEL WASTETANKS COMPARISON OF SRS AND HANFORDMODELING PREDICTIONS

    SciTech Connect

    Crawford, C; Ned Bibler, N

    2009-04-15

    In the high level waste tanks at the Savannah River Site (SRS), hydrogen is produced continuously by interaction of the radiation in the tank with water in the waste. Consequently, the vapor spaces of the tanks are purged to prevent the accumulation of H{sub 2} and possible formation of a flammable mixture in a tank. Personnel at SRS have developed an empirical model to predict the rate of H{sub 2} formation in a tank. The basis of this model is the prediction of the G value for H{sub 2} production. This G value is the number of H{sub 2} molecules produced per 100 eV of radiolytic energy absorbed by the waste. Based on experimental studies it was found that the G value for H{sub 2} production from beta radiation and from gamma radiation were essentially equal. The G value for H{sub 2} production from alpha radiation was somewhat higher. Thus, the model has two equations, one for beta/gamma radiation and one for alpha radiation. Experimental studies have also indicated that both G values are decreased by the presence of nitrate and nitrite ions in the waste. These are the main scavengers for the precursors of H{sub 2} in the waste; thus the equations that were developed predict G values for hydrogen production as a function of the concentrations of these two ions in waste. Knowing the beta/gamma and alpha heat loads in the waste allows one to predict the total generation rate for hydrogen in a tank. With this prediction a ventilation rate can be established for each tank to ensure that a flammable mixture is not formed in the vapor space in a tank. Recently personnel at Hanford have developed a slightly different model for predicting hydrogen G values. Their model includes the same precursor for H{sub 2} as the SRS model but also includes an additional precursor not in the SRS model. Including the second precursor for H{sub 2} leads to different empirical equations for predicting the G values for H{sub 2} as a function of the nitrate and nitrite concentrations in

  4. Hillslope inter-site comparisons to determine the controls on hillslope-stream connectivity and threshold catchment responses

    NASA Astrophysics Data System (ADS)

    van Meerveld, Ilja

    2015-04-01

    The relation between precipitation and streamflow is often characterized by a threshold, with runoff ratios or streamflow volumes being small during small events and dry conditions and increasing significantly during large events and wet conditions. These streamflow thresholds are frequently attributed to the addition of hillslope runoff to streamflow during large events and wet conditions and thus the hydrologic connection of hillslopes to the stream network. The addition of hillslope water to the stream can also lead to large changes in water quality. In areas where overland flow is uncommon, such as forested catchments in temperate climates, hillslope-stream connectivity requires the generation of lateral subsurface flow from hillslopes and a connection of saturated zones in hillslopes with each other and the riparian zone or the stream network. Most hillslope hydrological studies have focused on the subsurface flow and groundwater responses at individual research sites, rather than extracting more general rules and controls on how saturated zones in hillslopes connect with each other and the riparian zone or stream. Even for well-studied hillslopes, it is difficult to determine the main controls on the expansion of saturated areas and hillslope-stream connectivity because of our limited ability to observe flow pathways in the soil, the spatial variability in soil properties, the limited spatial scale of our measurements, temporal variability in antecedent moisture conditions and because datasets are usually short and do not include extreme events. Numerical simulation allows for systematic analyses of hillslope hydrological responses without requiring a large number of observations. However, most hillslope modelling studies so far are based on datasets from a few intensively studied hillslopes for which data is made publicly available, and are thus partly biased to these hillslopes. Inter-site comparisons have been shown to provide useful information on

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

    SciTech Connect

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

    2009-01-01

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

  6. Evaluation of physical activity web sites for use of behavior change theories.

    PubMed

    Doshi, Amol; Patrick, Kevin; Sallis, James F; Calfas, Karen

    2003-01-01

    Physical activity (PA) Web sites were assessed for their use of behavior change theories, including constructs of the health belief model, Transtheoretical Model, social cognitive theory, and the theory of reasoned action and planned behavior. An evaluation template for assessing PA Web sites was developed, and content validity and interrater reliability were demonstrated. Two independent raters evaluated 24 PA Web sites. Web sites varied widely in application of theory-based constructs, ranging from 5 to 48 on a 100-point scale. The most common intervention strategies were general information, social support, and realistic goal areas. Coverage of theory-based strategies was low, varying from 26% for social cognitive theory to 39% for health belief model. Overall, PA Web sites provided little assessment, feedback, or individually tailored assistance for users. They were unable to substantially tailor the on-line experience for users at different stages of change or different demographic characteristics.

  7. A comparison of classical and quantum analyses of electron localization sites in liquid water

    NASA Astrophysics Data System (ADS)

    Motakabbir, Kazi A.; Schnitker, Jurgen; Rossky, Peter J.

    1992-08-01

    The results of an earlier study [Schnitker, Rossky, and Kenney-Wallace, J. Chem. Phys. 85, 2986 (1986)] in which likely sites for electron localization in pure liquid water were identified and characterized via a physically motivated purely classical analysis are statistically compared to a corresponding fully quantum mechanical treatment of the excess electronic ground state. It is shown that the most energetically favorable localization sites identified by the classical treatment correspond reasonably to the quantum mechanical result both energetically and spatially. It is found that the existence and location of a physically localized ground state can be determined from the classical results if both the minimum of the estimated absolute total electronic energy and the difference between this minimum and the alternative local minima identified within a solvent configuration are considered. Further, the results confirm that the concentration of such effective sites is relatively high in the liquid (˜0.01 M). Hence, the classical approach has merit as a qualitative tool for the analysis of the electronic states supported by the preexisting configurational order in a liquid.

  8. Comparison of splice sites reveals that long noncoding RNAs are evolutionarily well conserved.

    PubMed

    Nitsche, Anne; Rose, Dominic; Fasold, Mario; Reiche, Kristin; Stadler, Peter F

    2015-05-01

    Large-scale RNA sequencing has revealed a large number of long mRNA-like transcripts (lncRNAs) that do not code for proteins. The evolutionary history of these lncRNAs has been notoriously hard to study systematically due to their low level of sequence conservation that precludes comprehensive homology-based surveys and makes them nearly impossible to align. An increasing number of special cases, however, has been shown to be at least as old as the vertebrate lineage. Here we use the conservation of splice sites to trace the evolution of lncRNAs. We show that >85% of the human GENCODE lncRNAs were already present at the divergence of placental mammals and many hundreds of these RNAs date back even further. Nevertheless, we observe a fast turnover of intron/exon structures. We conclude that lncRNA genes are evolutionary ancient components of vertebrate genomes that show an unexpected and unprecedented evolutionary plasticity. We offer a public web service (http://splicemap.bioinf.uni-leipzig.de) that allows to retrieve sets of orthologous splice sites and to produce overview maps of evolutionarily conserved splice sites for visualization and further analysis. An electronic supplement containing the ncRNA data sets used in this study is available at http://www.bioinf.uni-leipzig.de/publications/supplements/12-001.

  9. A Systematic Comparison of Teaching Hospital and Remote-Site Clinical Education

    ERIC Educational Resources Information Center

    And Others; Friedman, Charles P.

    1978-01-01

    A methodology is presented for examining activities of medical students on multisite clinical clerkships in obstetrics and gynecology. Five variables are explored: distribution of student activities, type or class of clinical conditions encountered, degree of "esoterism" of those conditions, type of student role, and flexibility of student role.…

  10. 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.

  11. Balance Comparisons between Female Dancers and Active Nondancers

    ERIC Educational Resources Information Center

    Ambegaonkar, Jatin P.; Caswell, Shane V.; Winchester, Jason B.; Shimokochi, Yohei; Cortes, Nelson; Caswell, Amanda M.

    2013-01-01

    Purpose: Female dancers have lower anterior cruciate ligament (ACL) injury rates compared with physically active women. Enhanced balance can decrease musculoskeletal injury risk. Dancers are proposed to have superior balance compared with physically active nondancers, and this may reduce their risk for ACL injury. However, whether female dancers…

  12. Active Ageing Level of Older Persons: Regional Comparison in Thailand.

    PubMed

    Haque, Md Nuruzzaman

    2016-01-01

    Active ageing level and its discrepancy in different regions (Bangkok, Central, North, Northeast, and South) of Thailand have been examined for prioritizing the policy agenda to be implemented. Attempt has been made to test preliminary active ageing models for Thai older persons and hence active ageing index (AAI, ranges from 0 to 1) has been estimated. Using nationally representative data and confirmatory factor analysis approach, this study justified active ageing models for female and male older persons in Thailand. Results revealed that active ageing level of Thai older persons is not high (mean AAIs for female and male older persons are 0.64 and 0.61, resp., and those are significantly different (p < 0.001)). Mean AAI in Central region is lower than North, Northeast, and South regions but there is no significant difference in the latter three regions of Thailand. Special emphasis should be given to Central region and policy should be undertaken for increasing active ageing level. Implementation of an Integrated Active Ageing Package (IAAP), containing policies for older persons to improve their health and economic security, to promote participation in social groups and longer working lives, and to arrange learning programs, would be helpful for increasing older persons' active ageing level in Thailand. PMID:27375903

  13. Active Ageing Level of Older Persons: Regional Comparison in Thailand.

    PubMed

    Haque, Md Nuruzzaman

    2016-01-01

    Active ageing level and its discrepancy in different regions (Bangkok, Central, North, Northeast, and South) of Thailand have been examined for prioritizing the policy agenda to be implemented. Attempt has been made to test preliminary active ageing models for Thai older persons and hence active ageing index (AAI, ranges from 0 to 1) has been estimated. Using nationally representative data and confirmatory factor analysis approach, this study justified active ageing models for female and male older persons in Thailand. Results revealed that active ageing level of Thai older persons is not high (mean AAIs for female and male older persons are 0.64 and 0.61, resp., and those are significantly different (p < 0.001)). Mean AAI in Central region is lower than North, Northeast, and South regions but there is no significant difference in the latter three regions of Thailand. Special emphasis should be given to Central region and policy should be undertaken for increasing active ageing level. Implementation of an Integrated Active Ageing Package (IAAP), containing policies for older persons to improve their health and economic security, to promote participation in social groups and longer working lives, and to arrange learning programs, would be helpful for increasing older persons' active ageing level in Thailand.

  14. Universities and Economic Development Activities: A UK Regional Comparison

    ERIC Educational Resources Information Center

    Decter, Moira; Cave, Frank; Rose, Mary; Peers, Gill; Fogg, Helen; Smith, Susan M.

    2011-01-01

    A number of UK universities prioritize economic development or regeneration activities and for some of these universities such activities are the main focus of their knowledge transfer work. This study compares two regions of the UK--the North West and the South East of England--which have very different levels of economic performance.…

  15. 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.

  16. Can we define an asymptotic value for the ice active surface site density for heterogeneous ice nucleation?

    NASA Astrophysics Data System (ADS)

    Niedermeier, Dennis; Augustin-Bauditz, Stefanie; Hartmann, Susan; Wex, Heike; Ignatius, Karoliina; Stratmann, Frank

    2015-05-01

    The immersion freezing behavior of droplets containing size-segregated, monodisperse feldspar particles was investigated. For all particle sizes investigated, a leveling off of the frozen droplet fraction was observed reaching a plateau within the heterogeneous freezing temperature regime (T >- 38°C). The frozen fraction in the plateau region was proportional to the particle surface area. Based on these findings, an asymptotic value for ice active surface site density ns, which we named ns⋆, could be determined for the investigated feldspar sample. The comparison of these results with those of other studies not only elucidates the general feasibility of determining such an asymptotic value but also shows that the value of ns⋆ strongly depends on the method of the particle surface area determination. However, such an asymptotic value might be an important input parameter for atmospheric modeling applications. At least it shows that care should be taken when ns is extrapolated to lower or higher temperature.

  17. A conformational switch in the active site of BT_2972, a methyltransferase from an antibiotic resistant pathogen B. thetaiotaomicron.

    PubMed

    Kumar, Veerendra; Sivaraman, J

    2011-01-01

    Methylation is one of the most common biochemical reactions involved in cellular and metabolic functions and is catalysed by the action of methyltransferases. Bacteroides thetaiotaomicron is an antibiotic-resistant bacterium that confers resistance through methylation, and as yet, there is no report on the structure of methyltransferases from this bacterium. Here, we report the crystal structure of an AdoMet-dependent methyltransferase, BT_2972 and its complex with AdoMet and AdoHcy for B. thetaiotaomicron VPI-5482 strain along with isothermal titration calorimetric assessment of the binding affinities. Comparison of the apo and complexed BT_2972 structures reveals a significant conformational change between open and closed forms of the active site that presumably regulates the association with cofactors and may aid interaction with substrate. Together, our analysis suggests that BT_2972 is a small molecule methyltransferase and might catalyze two O-methylation reaction steps involved in the ubiquinone biosynthesis pathway. PMID:22140448

  18. Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

    PubMed Central

    Tomkinson, B; Wernstedt, C; Hellman, U; Zetterqvist, O

    1987-01-01

    The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases. PMID:3313395

  19. Evolution of anatase surface active sites probed by in situ sum-frequency phonon spectroscopy

    PubMed Central

    Cao, Yue; Chen, Shiyou; Li, Yadong; Gao, Yi; Yang, Deheng; Shen, Yuen Ron; Liu, Wei-Tao

    2016-01-01

    Surface active sites of crystals often govern their relevant surface chemistry, yet to monitor them in situ in real atmosphere remains a challenge. Using surface-specific sum-frequency spectroscopy, we identified the surface phonon mode associated with the active sites of undercoordinated titanium ions and conjoint oxygen vacancies, and used it to monitor them on anatase (TiO2) (101) under ambient conditions. In conjunction with theory, we determined related surface structure around the active sites and tracked the evolution of oxygen vacancies under ultraviolet irradiation. We further found that unlike in vacuum, the surface oxygen vacancies, which dominate the surface reactivity, are strongly regulated by ambient gas molecules, including methanol and water, as well as weakly associated species, such as nitrogen and hydrogen. The result revealed a rich interplay between prevailing ambient species and surface reactivity, which can be omnipresent in environmental and catalytic applications of titanium dioxides. PMID:27704049

  20. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase.

    PubMed

    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.

  1. Solvent Tuning of Electrochemical Potentials in the Active Sites of HiPIP Versus Ferredoxin

    SciTech Connect

    Dey, A.; Francis, E.J.; Adams, M.W.W.; Babini, E.; Takahashi, Y.; Fukuyama, K.; Hodgson, K.O.; Hedman, B.; Solomon, E.I.; /Stanford U., Chem. Dept. /Georgia U. /Bologna U. /Osaka U. /SLAC, SSRL

    2009-04-29

    A persistent puzzle in the field of biological electron transfer is the conserved iron-sulfur cluster motif in both high potential iron-sulfur protein (HiPIP) and ferredoxin (Fd) active sites. Despite this structural similarity, HiPIPs react oxidatively at physiological potentials, whereas Fds are reduced. Sulfur K-edge x-ray absorption spectroscopy uncovers the substantial influence of hydration on this variation in reactivity. Fe-S covalency is much lower in natively hydrated Fd active sites than in HiPIPs but increases upon water removal; similarly, HiPIP covalency decreases when unfolding exposes an otherwise hydrophobically shielded active site to water. Studies on model compounds and accompanying density functional theory calculations support a correlation of Fe-S covalency with ease of oxidation and therefore suggest that hydration accounts for most of the difference between Fd and HiPIP reduction potentials.

  2. 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.

  3. Wasp recruitment to the T cell:APC contact site occurs independently of Cdc42 activation.

    PubMed

    Cannon, J L; Labno, C M; Bosco, G; Seth, A; McGavin, M H; Siminovitch, K A; Rosen, M K; Burkhardt, J K

    2001-08-01

    Cdc42 and WASP are critical regulators of actin polymerization whose function during T cell signaling is poorly understood. Using a novel reagent that specifically detects Cdc42-GTP in fixed cells, we found that activated Cdc42 localizes to the T cell:APC contact site in an antigen-dependent manner. TCR signaling alone was sufficient to induce localization of Cdc42-GTP, and functional Lck and Zap-70 kinases were required. WASP also localized to the T cell:APC contact site in an antigen-dependent manner. Surprisingly, WASP localization was independent of the Cdc42 binding domain but required the proline-rich domain. Our results indicate that localized WASP activation requires the integration of multiple signals: WASP is recruited via interaction with SH3 domain-containing proteins and is activated by Cdc42-GTP concentrated at the same site. PMID:11520460

  4. 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

  5. Comparison of air dispersion modeling results with ambient air sampling data: A case study at Tacoma Landfill, a National Priorities List Site

    SciTech Connect

    Griffin, L.R. ); Rutherford, T.L. )

    1994-08-01

    Air dispersion modeling, ambient air sampling, and emissions testing of landfill sources have been performed to evaluate the effects of remedial activities on ambient air surrounding the Tacoma Landfill. In 1983, the Tacoma Landfill was placed on the National Priorities List (NPL) as part of the Commencement Bay/South Tacoma Channel Superfund site. Remedial activities completed, or near completion, at the 190 acre (768,903 m[sup 2]) Tacoma Landfill include a groundwater extraction system and air stripping units used to remove volatile organic compounds (VOCs) from groundwater, landfill gas extraction and flare system to control gas migration from the landfill, landfill liner and leachate collection system for an active section of the landfill, and a landfill cap that covers the inactive portions of the landfill. Dispersion modeling was performed with measured stack emission data using Industrial Source Complex (ISC) to determine the groundlevel concentrations of VOCs from the air stripper, flares, and active portion of the landfill for comparison with the measured ambient air data collected during 1992. 9 refs., 3 figs., 6 tabs.

  6. Comparison of modification sites in glycated crystallin in vitro and in vivo.

    PubMed

    Kielmas, Martyna; Kijewska, Monika; Kluczyk, Alicja; Oficjalska, Jolanta; Gołębiewska, Bożena; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2015-03-01

    Glycation of α-crystallin is responsible for age- and diabetic-related cataracts, which are the main cause of blindness worldwide. We optimized the method of identification of lysine residues prone to glycation using the combination of LC-MS, isotopic labeling, and modified synthetic peptide standards with the glycated lysine derivative (Fmoc-Lys(i,i-Fru,Boc)-OH). The in vitro glycation of bovine lens α-crystallin was conducted by optimized method with the equimolar mixture of [(12)C6]- and [(13)C6]D-glucose. The in vivo glycation was studied on human lens crystallin. The glycated protein was subjected to proteolysis and analyzed using LC-MS. The results of in vitro and in vivo glycation of α-crystallin reveal a different distribution of the modified lysine residues. More Amadori products were detected as a result of the in vitro reaction due to forced glycation conditions. The developed method allowed us to identify the glycation sites in crystallin from eye lenses obtained from patients suffering from the cataract. We identified K166 in the A chain and K166 in the B chain of α-crystallin as major glycation sites during the in vitro reaction. We found also two in vivo glycated lysine residues: K92 in the B chain and K166 in the A chain, which are known as locations for Amadori products. These modification sites were confirmed by the LC-MS experiment using two synthetic standards. This study demonstrates the applicability of the LC-MS methods combined with the isotopic labeling and synthetic peptide standards for analysis of post-translational modifications in the biological material. PMID:25636230

  7. 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.

  8. Comparison of total mercury and methylmercury cycling at five sites using the small watershed approach

    USGS Publications Warehouse

    Shanley, J.B.; Alisa, Mast M.; Campbell, D.H.; Aiken, G.R.; Krabbenhoft, D.P.; Hunt, R.J.; Walker, J.F.; Schuster, P.F.; Chalmers, A.; Aulenbach, Brent T.; Peters, N.E.; Marvin-DiPasquale, M.; Clow, D.W.; Shafer, M.M.

    2008-01-01

    The small watershed approach is well-suited but underutilized in mercury research. We applied the small watershed approach to investigate total mercury (THg) and methylmercury (MeHg) dynamics in streamwater at the five diverse forested headwater catchments of the US Geological Survey Water, Energy, and Biogeochemical Budgets (WEBB) program. At all sites, baseflow THg was generally less than 1 ng L-1 and MeHg was less than 0.2 ng L-1. THg and MeHg concentrations increased with streamflow, so export was primarily episodic. At three sites, THg and MeHg concentration and export were dominated by the particulate fraction in association with POC at high flows, with maximum THg (MeHg) concentrations of 94 (2.56) ng L-1 at Sleepers River, Vermont; 112 (0.75) ng L-1 at Rio Icacos, Puerto Rico; and 55 (0.80) ng L-1 at Panola Mt., Georgia. Filtered (<0.7 ??m) THg increased more modestly with flow in association with the hydrophobic acid fraction (HPOA) of DOC, with maximum filtered THg concentrations near 5 ng L-1 at both Sleepers and Icacos. At Andrews Creek, Colorado, THg export was also episodic but was dominated by filtered THg, as POC concentrations were low. MeHg typically tracked THg so that each site had a fairly constant MeHg/THg ratio, which ranged from near zero at Andrews to 15% at the low-relief, groundwater-dominated Allequash Creek, Wisconsin. Allequash was the only site with filtered MeHg consistently above detection, and the filtered fraction dominated both THg and MeHg. Relative to inputs in wet deposition, watershed retention of THg (minus any subsequent volatilization) was 96.6% at Allequash, 60% at Sleepers, and 83% at Andrews. Icacos had a net export of THg, possibly due to historic gold mining or frequent disturbance from landslides. Quantification and interpretation of Hg dynamics was facilitated by the small watershed approach with emphasis on event sampling. ?? 2008 Elsevier Ltd. All rights reserved.

  9. Comparison of total mercury and methylmercury cycling at five sites using the small watershed approach.

    PubMed

    Shanley, James B; Alisa Mast, M; Campbell, Donald H; Aiken, George R; Krabbenhoft, David P; Hunt, Randall J; Walker, John F; Schuster, Paul F; Chalmers, Ann; Aulenbach, Brent T; Peters, Norman E; Marvin-DiPasquale, Mark; Clow, David W; Shafer, Martin M

    2008-07-01

    The small watershed approach is well-suited but underutilized in mercury research. We applied the small watershed approach to investigate total mercury (THg) and methylmercury (MeHg) dynamics in streamwater at the five diverse forested headwater catchments of the US Geological Survey Water, Energy, and Biogeochemical Budgets (WEBB) program. At all sites, baseflow THg was generally less than 1ng L(-1) and MeHg was less than 0.2ng L(-1). THg and MeHg concentrations increased with streamflow, so export was primarily episodic. At three sites, THg and MeHg concentration and export were dominated by the particulate fraction in association with POC at high flows, with maximum THg (MeHg) concentrations of 94 (2.56)ng L(-1) at Sleepers River, Vermont; 112 (0.75)ng L(-1) at Rio Icacos, Puerto Rico; and 55 (0.80)ng L(-1) at Panola Mt., Georgia. Filtered (<0.7microm) THg increased more modestly with flow in association with the hydrophobic acid fraction (HPOA) of DOC, with maximum filtered THg concentrations near 5ng L(-1) at both Sleepers and Icacos. At Andrews Creek, Colorado, THg export was also episodic but was dominated by filtered THg, as POC concentrations were low. MeHg typically tracked THg so that each site had a fairly constant MeHg/THg ratio, which ranged from near zero at Andrews to 15% at the low-relief, groundwater-dominated Allequash Creek, Wisconsin. Allequash was the only site with filtered MeHg consistently above detection, and the filtered fraction dominated both THg and MeHg. Relative to inputs in wet deposition, watershed retention of THg (minus any subsequent volatilization) was 96.6% at Allequash, 60% at Sleepers, and 83% at Andrews. Icacos had a net export of THg, possibly due to historic gold mining or frequent disturbance from landslides. Quantification and interpretation of Hg dynamics was facilitated by the small watershed approach with emphasis on event sampling.

  10. 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

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

    NASA Astrophysics Data System (ADS)

    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.

    2016-08-01

    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.

  12. 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.

  13. SABER: A computational method for identifying active sites for new reactions

    PubMed Central

    Nosrati, Geoffrey R; Houk, K N

    2012-01-01

    A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644–1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were l-Ala d/l-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. PMID:22492397

  14. SABER: a computational method for identifying active sites for new reactions.

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-05-01

    A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644-1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were L-Ala D/L-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. PMID:22492397

  15. Comparison of Estimated and Measured Muscle Activity During Inclined Walking.

    PubMed

    Alexander, Nathalie; Schwameder, Hermann

    2016-04-01

    While inclined walking is a frequent daily activity, muscle forces during this activity have rarely been examined. Musculoskeletal models are commonly used to estimate internal forces in healthy populations, but these require a priori validation. The aim of this study was to compare estimated muscle activity using a musculoskeletal model with measured EMG data during inclined walking. Ten healthy male participants walked at different inclinations of 0°, ± 6°, ± 12°, and ± 18° on a ramp equipped with 2 force plates. Kinematics, kinetics, and muscle activity of the musculus (m.) biceps femoris, m. rectus femoris, m. vastus lateralis, m. tibialis anterior, and m. gastrocnemius lateralis were recorded. Agreement between estimated and measured muscle activity was determined via correlation coefficients, mean absolute errors, and trend analysis. Correlation coefficients between estimated and measured muscle activity for approximately 69% of the conditions were above 0.7. Mean absolute errors were rather high with only approximately 38% being ≤ 30%. Trend analysis revealed similar estimated and measured muscle activities for all muscles and tasks (uphill and downhill walking), except m. tibialis anterior during uphill walking. This model can be used for further analysis in similar groups of participants.

  16. 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.

  17. 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.

  18. 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

  19. Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

    SciTech Connect

    Patel, Asmita; Shuman, Stewart; Mondragon, Alfonso

    2010-03-08

    Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-{angstrom} crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) {beta}-sheet domain (amino acids 1-90) and a predominantly {alpha}-helical carboxyl-terminal (C) domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an 'open' conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.

  20. Comparison of pesticide leaching potential to groundwater under EU FOCUS and site specific conditions.

    PubMed

    Labite, Herve; Holden, Nicholas M; Richards, Karl G; Kramers, Gaelene; Premrov, Alina; Coxon, Catherine E; Cummins, Enda

    2013-10-01

    The EU FOCUS scenarios are a set of nine standard scenarios based on a combination of crop, soil and weather data used throughout Europe to evaluate the leaching potential of pesticides to groundwater. In Ireland, two predefined EU FOCUS scenarios (Okehampton and Hamburg) appear to be the most appropriate to Irish conditions. However, there is concern that these scenarios may not accurately represent Irish specific conditions, especially in terms of soil and climatic weather. Therefore, the objective of this study was to parameterise a number of site specific locations in Ireland (represented by Oakpark, Clonroche, Rathangan and Elton series soils) and to compare simulated leachate levels at these locations to EU FOCUS scenarios using the PELMO (Pesticide Leaching Model) simulation model. The hydrological processes were validated using observed data for soil tension and leachate. The appropriate EU FOCUS scenarios were then simulated for the given locations and compared to the parameterised scenario. All scenarios were run using the same version of PELMO, therefore eliminating any software impacts. The models were run for 26 years using appropriate meteorological data. The results showed significant difference between the parameterised model pesticide leaching and that resulting from the EU FOCUS scenarios, the latter overestimating site pesticide leaching from 42 to 99%. The results indicated a significant conservatism in using EU FOCUS scenarios to determine potential pesticide concentration in the leachate under Irish specific conditions and ensure the desired level of protection against pesticide contamination of national water resources.

  1. Comparison of environmental issues related to development of small hydropower resources at new versus existing sites

    SciTech Connect

    Loar, J. M.; Hildebrand, S. G.

    1980-01-01

    Many of the ecological issues associated with the development of small hydropower resources are similar at both new (undeveloped) sites and those with existing dams that will be retrofitted for hydroelectric generation. Issues that could occur with both types of development are: (1) blockage of fish migration routes; (2) water level fluctuations; (3) instream flows, (4) water quality; (5) dredging and dredged material disposal; and (6) threatened or endangered species. However, new site development projects require the alteration of existing aquatic and terrestrial ecosystems that will be, in most cases, significantly greater than the environmental changes associated with the retrofitting of existing dams. Although project design and operation are important factors controlling the nature and magnitude of the environmental impacts of small hydropower resource development, the mitigation of adverse impacts (and the optimization of beneficial effects) is dependent, in large measure, on our ability to accurately predict physical, chemical, and biological changes. Predicting the impacts of new impoundments may be considerably more difficult than predicting the impacts that might occur if an existing dam/impoundment system is developed. A comparative approach at the ecosystem level can provide valuable insights into the structure and function of reservoir systems and significantly increase our predictive capability.

  2. Comparison of shear-wave slowness profiles at 10 strong-motion sites from noninvasive SASW measurements and measurements made in boreholes

    USGS Publications Warehouse

    Brown, L.T.; Boore, D.M.; Stokoe, K.H.

    2002-01-01

    The spectral-analysis-of-surface-waves (SASW) method is a relatively new in situ method for determining shear-wave slownesses. All measurements are made on the ground surface, making it much less costly than methods that require boreholes. The SASW method uses a number of active sources (ranging from a commercial Vibroseis truck to a small handheld hammer for the study conducted here) and different receiver spacings to map a curve of apparent phase velocity versus frequency. With the simplifying assumption that the phase velocities correspond to fundamental mode surface waves, forward modeling yields an estimate of the sub-surface shear-wave slownesses. To establish the reliability of this indirect technique, we conducted a blind evaluation of the SASW method. SASW testing was performed at 10 strong-motion stations at which borehole seismic measurements were previously or subsequently made; if previously made, the borehole results were not used for the interpretation of the SASW data, and vice-versa. Comparisons of the shear-wave slownesses from the SASW and borehole measurements are generally very good. The differences in predicted ground-motion amplifications are less than about 15% for most frequencies. In addition, both methods gave the same NEHRP site classification for seven of the sites. For the other three sites the average velocities from the downhole measurements were only 5-13 m/sec larger than the velocity defining the class C/D boundary. This study demonstrates that in many situations the SASW method can provide subsurface information suitable for site response predictions.

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. Analysis of active site residues of the antiviral protein from summer leaves from Phytolacca americana by site-directed mutagenesis.

    PubMed

    Poyet, J L; Hoeveler, A; Jongeneel, C V

    1998-12-30

    The summer leaf isoform of the pokeweed (Phytolacca americana) antiviral protein, PAP II, was produced in high yields from inclusion bodies in recombinant E. coli. On the basis of its sequence similarity with the spring leaf isoform (PAP I) and with the A chain of ricin, a three-dimensional model of the protein was constructed as an aid in the design of active site mutants. PAP II variants mutated in residues Asp 88 (D88N), Tyr 117 (Y117S), Glu 172 (E172Q), Arg 175 (R175H) and a combination of Asp 88 and Arg 175 (D88N/R175H) were produced in E. coli and assayed for their ability to inhibit protein synthesis in a rabbit reticulocyte lysate. All of these mutations had effects deleterious to the enzymatic activity of PAP II. The results were interpreted in the light of three reaction mechanisms proposed for ribosome-inactivating proteins (RIPs). We conclude that none of the proposed mechanisms is entirely consistent with the data presented here.

  9. Aerosol Property Comparison Within and Above the ABL at the ARM Program SGP Site

    SciTech Connect

    Delle Monache, L

    2002-05-01

    This thesis determines what, if any, measurements of aerosol properties made at the Earth surface are representative of those within the entire air column. Data from the Atmospheric Radiation Measurement site at the Southern Great Plains, the only location in the world where ground-based and in situ airborne measurements are routinely made. Flight legs during the one-year period from March 2000 were categorized as either within or above the atmospheric boundary layer (ABL) by use of an objective mixing height determination technique. Correlations between aerosol properties measured at the surface and those within and above the ABL were computed. Aerosol extensive and intensive properties measured at the surface were found representative of values within the ABL, but not of within the free atmosphere.

  10. Comparison of the non-proliferation event aftershocks with other Nevada Test Site events

    SciTech Connect

    Jarpe, S.; Goldstein, P.; Zucca, J.J.

    1994-04-01

    As part of a larger effort to develop technology for on-site inspection of ambiguous underground seismic events, we have been working to identify phenomenology of aftershock seismicity which would be useful for discriminating between nuclear explosions, chemical explosions, earthquakes or other seismic events. Phenomenology we have investigated includes; the spatial distribution of aftershocks, the number of aftershocks as a function of time after the main event, the size of the aftershocks, and waveform frequency content. Our major conclusions are: (1) Depending on local geologic conditions, aftershock production rate two weeks after zero time ranges from 1 to 100 per day. (2) Aftershocks of concentrated chemical explosions such as the NPE are indistinguishable from aftershocks of nuclear explosions. (3) Earthquake and explosion aftershock sequences may be differentiated on the basis of depth, magnitude, and in some cases, frequency content of seismic signals.

  11. A comparison of actual and perceived residential proximity to toxic waste sites.

    PubMed

    Howe, H L

    1988-01-01

    Studies of Memphis and Three Mile Island have noted a positive association between actual residential distance and public concern about exposure to the potential of contamination, whereas none was found at Love Canal. In this study, concern about environmental contamination and exposure was examined in relation to both perceived and actual proximity to a toxic waste disposal site (TWDS). It was hypothesized that perceived residential proximity would better predict concern levels that would actual residential distance. The data were abstracted from a New York State, excluding New York City, survey using all respondents (N = 317) from one county known to have a large number of TWDSs. Using linear regression, the variance explained in concern scores was 22 times higher with perceived distance than for actual distance. Perceived residential distance was a significant predictor of concern scores, while actual distance was not. However, perceived distance explained less than 5% of the variance in concern scores. PMID:3196077

  12. Comparison of Kriging and coKriging for soil contamination mapping in abandoned mine sites

    NASA Astrophysics Data System (ADS)

    Lee, Hyeongyu; Choi, Yosoon

    2015-04-01

    Soil contamination mapping around abandoned mines is an important task for the planning and design of mine reclamation. This study compared the ordinary Kriging and the co-Kriging methods for the soil contamination mapping in abandoned mine sites. Four approaches were conducted as follows: (1) soil contamination mapping using the ordinary Kriging and Inductively Coupled Plasma (ICP) data only; (2) soil contamination mapping using the ordinary Kriging and Portable X-Ray Fluorescence (PXRF) data only; (3) soil contamination mapping using the ordinary Kriging and integrated data from ICP and PXRF; and (4) soil contamination mapping using the co-Kriging and integrated data from ICP and PXRF. Results indicate that the approach 3 provides substantial improvements over other three approaches including a more reasonable spatial pattern of soil contamination and reduction in the error of its estimates.

  13. 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.

  14. Maintenance of plastid RNA editing activities independently of their target sites

    PubMed Central

    Tillich, Michael; Poltnigg, Peter; Kushnir, Sergei; Schmitz-Linneweber, Christian

    2006-01-01

    RNA editing in plant organelles is mediated by site-specific, nuclear-encoded factors. Previous data suggested that the maintenance of these factors depends on the presence of their rapidly evolving cognate sites. The surprising ability of allotetraploid Nicotiana tabacum (tobacco) to edit a foreign site in the chloroplast ndhA messenger RNA was thought to be inherited from its diploid male ancestor, Nicotiana tomentosiformis. Here, we show that the same ndhA editing activity is also present in Nicotiana sylvestris, which is the female diploid progenitor of tobacco and which lacks the ndhA site. Hence, heterologous editing is not simply a result of tobacco's allopolyploid genome organization. Analyses of other editing sites after sexual or somatic transfer between land plants showed that heterologous editing occurs at a surprisingly high frequency. This suggests that the corresponding editing activities are conserved despite the absence of their target sites, potentially because they serve other functions in the plant cell. PMID:16415790

  15. Engelmann Spruce Site Index Models: A Comparison of Model Functions and Parameterizations

    PubMed Central

    Nigh, Gordon

    2015-01-01

    Engelmann spruce (Picea engelmannii Parry ex Engelm.) is a high-elevation species found in western Canada and western USA. As this species becomes increasingly targeted for harvesting, better height growth information is required for good management of this species. This project was initiated to fill this need. The objective of the project was threefold: develop a site index model for Engelmann spruce; compare the fits and modelling and application issues between three model formulations and four parameterizations; and more closely examine the grounded-Generalized Algebraic Difference Approach (g-GADA) model parameterization. The model fitting data consisted of 84 stem analyzed Engelmann spruce site trees sampled across the Engelmann Spruce – Subalpine Fir biogeoclimatic zone. The fitted models were based on the Chapman-Richards function, a modified Hossfeld IV function, and the Schumacher function. The model parameterizations that were tested are indicator variables, mixed-effects, GADA, and g-GADA. Model evaluation was based on the finite-sample corrected version of Akaike’s Information Criteria and the estimated variance. Model parameterization had more of an influence on the fit than did model formulation, with the indicator variable method providing the best fit, followed by the mixed-effects modelling (9% increase in the variance for the Chapman-Richards and Schumacher formulations over the indicator variable parameterization), g-GADA (optimal approach) (335% increase in the variance), and the GADA/g-GADA (with the GADA parameterization) (346% increase in the variance). Factors related to the application of the model must be considered when selecting the model for use as the best fitting methods have the most barriers in their application in terms of data and software requirements. PMID:25853472

  16. Comparison of high-frequency seismic sources at the Grimsel test site, central Alps, Switzerland

    SciTech Connect

    Buehnemann, J.; Holliger, K.

    1998-07-01

    In August 1995, various high-frequency seismic sources were tested at the Grimsel Test Site (GTS), located inside a crystalline rock body in the central Swiss Alps. These source tests were designed to facilitate future tomographic studies of potential radioactive waste disposal sites. The principal objective was to identify borehole and tunnel seismic sources capable of generating powerful high-frequency signals such that frequencies up to 1,000 Hz can be observed over distances of 1,000 m in crystalline or consolidated sedimentary rocks. Seismic sources were situated in water-filled boreholes (sparker, two piezoelectric sources, explosives) and at or near the tunnel wall (accelerated weight drop, minivibrator, bolt gun, buffalo gun, explosives). To evaluate and compare the source characteristics, the direct P-wave generated by the various seismic sources was investigated for the decay of its S/N and dominant frequency with offset and for the maximum distance at which first arrivals could be picked. Of the seismic sources tested, small explosive charges (5--100 g) had the most favorable S/N and frequency characteristics. At GTS, the target distance ({approximately}1,000 m) was reached with explosive charges of 50 g or more. None of the sources tested was capable of generating signals that sustained frequencies of 1,000 Hz over distances in excess of 100 to 200 m. The unusually strong attenuation implied by this observation is likely due to the fact that the rocks at GTS underwent brittle deformation during the Alpine orogeny and therefore contain numerous fractures and shear zones.

  17. Phenotypic and Genotypic Comparison of Symbiotic and Free-Living Cyanobacteria from a Single Field Site

    PubMed Central

    West, N. J.; Adams, D. G.

    1997-01-01

    PCR amplification techniques were used to compare cyanobacterial symbionts from a cyanobacterium-bryophyte symbiosis and free-living cyanobacteria from the same field site. Thirty-one symbiotic cyanobacteria were isolated from the hornwort Phaeoceros sp. at several closely spaced locations, and 40 free-living cyanobacteria were isolated from the immediate vicinity of the same plants. One of the symbiotic isolates was a species of Calothrix, a genus not previously known to form bryophyte symbioses, and the remainder were Nostoc spp. Of the free-living strains, two were Calothrix spp., three were Chlorogloeopsis spp. and the rest were Nostoc spp. All of the symbiotic and all but one of the free-living strains were able to reconstitute the symbiosis with axenic cultures of both Phaeoceros and the liverwort Blasia sp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the regions flanking the 16S-23S rRNA internal transcribed spacer. With one exception, the two techniques produced complementary results and confirmed for the first time that a diversity of symbiotic cyanobacteria infect Phaeoceros in the field. Symbionts from adjacent colonies were different as often as they were the same, showing that the same thallus could be infected with many different cyanobacterial strains. Strains found to be identical by the techniques employed here were often found as symbionts in different thalli at the same locale but were never found free-living. Only one of the free-living strains, and none of the symbiotic strains, was found at more than one sample site, implying a highly localized distribution of strains. PMID:16535734

  18. Comparison of atmospheric pesticide concentrations measured at three sampling sites: local, regional and long-range transport.

    PubMed

    Sanusi, A; Millet, M; Mirabe, P; Wortham, H

    2000-12-18

    We present the comparison of atmospheric concentrations of eleven currently used pesticides (HCB, alpha-HCH, gamma-HCH, trifluraline, mecoprop, phosalone, atrazine, carbofuran, carbaryl, diuron and isoproturon) measured in remote (Aubure), rural (Colmar) and urban (Strasbourg) areas of Alsace and Vosges regions (cast France). Pesticides samples were collected simultaneously on two of the three sites during the summer season of 1993 and 1994, using a Hi-Vol sampler with Whatman filter paper and XAD-2 resin. The particle and gas phases were collected separately during 24 h. The relative importance of local emissions and local, regional and long distance transport on the contamination of the atmosphere in the three environments (remote, rural and urban) were investigated. To facilitate the interpretation of the results, the alpha/gamma-HCH ratio was used as a tracer of pesticide emissions.

  19. 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.

  20. Newly identified essential amino acid residues affecting Δ8-sphingolipid desaturase activity revealed by site-directed mutagenesis.

    PubMed

    Li, Shu-Fen; Song, Li-Ying; Zhang, Guo-Jun; Yin, Wei-Bo; Chen, Yu-Hong; Wang, Richard R-C; Hu, Zan-Min

    2011-12-01

    In order to identify amino acid residues crucial for the enzymatic activity of Δ(8)-sphingolipid desaturases, a sequence comparison was performed among Δ(8)-sphingolipid desaturases and Δ(6)-fatty acid desaturases from various plants. In addition to the known conserved cytb(5) (cytochrome b(5)) HPGG motif and three conserved histidine boxes, they share additional 15 completely conserved residues. A series of site-directed mutants were generated using our previously isolated Δ(8)-sphingolipid desaturase gene from Brassica rapa to evaluate the importance of these residues to the enzyme function. The mutants were functionally characterized by heterologous expression in yeast, allowing the identification of the products of the enzymes. The results revealed that residues H63, N203, D208, D210, and G368 were obligatorily required for the enzymatic activity, and substitution of the residues F59, W190, W345, L369 and Q372 markedly decreased the enzyme activity. Among them, replacement of the residues W190, L369 and Q372 also has significant influence on the ratio of the two enzyme products. Information obtained in this work provides the molecular basis for the Δ(8)-sphingolipid desaturase activity and aids in our understanding of the structure-function relationships of the membrane-bound desaturases.

  1. 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

  2. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions.

    PubMed

    Kelly, Paul P; Eichler, Anja; Herter, Susanne; Kranz, David C; Turner, Nicholas J; Flitsch, Sabine L

    2015-01-01

    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.

  3. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions.

    PubMed

    Kelly, Paul P; Eichler, Anja; Herter, Susanne; Kranz, David C; Turner, Nicholas J; Flitsch, Sabine L

    2015-01-01

    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

  4. Comparison of activation effects in {gamma}-ray detector materials

    SciTech Connect

    Truscott, P.R.; Evans, H.E.; Dyer, C.S.; Peerless, C.L.; Flatman, J.C.; Cosby, M.; Knight, P.; Moss, C.E.

    1996-06-01

    Activation induced by cosmic and trapped radiation in {gamma}-ray detector materials represents a significant source of background for space-based detector systems. Selection of detector materials should therefore include consideration of this background source. Results are presented from measurements of induced radioactivity in different scintillators activated either as a result of irradiation by mono-energetic protons at accelerator facilities, or flight on board the Space Shuttle. Radiation transport computer codes are used to help compare the effects observed from the scintillators, by identifying and quantifying the influence on the background spectra from more than one hundred of the radionuclides produced by spallation. For the space experiment data, the simulation results also permit determination of the contributions to detector activation from the different sources of radiation in the Shuttle cabin.

  5. 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

  6. Comparison of cytotoxic activities of extracts from Selaginella species

    PubMed Central

    Li, Juan; Lei, Xiang; Chen, Ke-li

    2014-01-01

    Background: Selaginella species are resurrection plants, which are known, possess various molecular bioactivities depending on species, but only a few species have been detailed observe in the advanced research. Objective: The objective of the following study is to compare the chemical profiles of different species of Selaginella and to investigate cytotoxicity and induction of apoptosis activities of some species of Selaginella. Materials and Methods: The high-performance liquid chromatography (HPLC) method was developed for chemical analysis. Ethyl acetate, ethanol and water-soluble extracts from seven Selaginella species were submitted to 3-(4,5-dimenthyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide assay, flow cytometry, deoxyribonucleic acid (DNA) laddering analysis and caspase-3 expression using Bel-7402, HT-29 and HeLa cells. Results: The HPLC analysis revealed two major common peaks, which were identified as amentoflavone and robustaflavone and another three main peaks in their chromatograms. The results showed that S. labordei, Selaginella tamariscina and Selaginella uncinata had relatively stronger activities on Bel-7402 and HeLa cells and Selaginella moellendorfii had moderate antiproliferation activities, but Selaginella remotifolia and Selaginella pulvinata had almost no inhibitory activities. The main active components were in the ethyl acetate extracts which had abundant biflavonoids. The effects of these extracts on cell proliferation and apoptosis in different cells were not the same, they were more apparent on HeLa cells than on HT-29 cells. The assay of DNA laddering analysis and caspase-3 expression further confirmed that inducing cell apoptosis was one of antitumor mechanisms and antitumor activities of Selaginella species were related to apoptosis induced by caspase family. Conclusion: S. labordei, S. tamariscina and S. uncinata would be potential antitumor agents. PMID:25422557

  7. A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon.

    PubMed

    Rashid, N; Morikawa, M; Kanaya, S; Atomi, H; Imanaka, T

    1999-02-19

    A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein.

  8. Docking and molecular dynamics studies at trypanothione reductase and glutathione reductase active sites.

    PubMed

    Iribarne, Federico; Paulino, Margot; Aguilera, Sara; Murphy, Miguel; Tapia, Orlando

    2002-05-01

    A theoretical docking study on the active sites of trypanothione reductase (TR) and glutathione reductase (GR) with the corresponding natural substrates, trypanothione disulfide (T[S]2) and glutathione disulfide (GSSG), is reported. Molecular dynamics simulations were carried out in order to check the robustness of the docking results. The energetic results are in agreement with previous experimental findings and show the crossed complexes have lower stabilization energies than the natural ones. To test DOCK3.5, four nitro furanic compounds, previously designed as potentially active anti-chagasic molecules, were docked at the GR and TR active sites with the DOCK3.5 procedure. A good correlation was found between differential inhibitory activity and relative interaction energy (affinity). The results provide a validation test for the use of DOCK3.5 in connection with the design of anti-chagasic drugs.

  9. Active sites and mechanisms for direct oxidation of benzene to phenol over carbon catalysts.

    PubMed

    Wen, Guodong; Wu, Shuchang; Li, Bo; Dai, Chunli; Su, Dang Sheng

    2015-03-23

    The direct oxidation of benzene to phenol with H2 O2 as the oxidizer, which is regarded as an environmentally friendly process, can be efficiently catalyzed by carbon catalysts. However, the detailed roles of carbon catalysts, especially what is the active site, are still a topic of debate controversy. Herein, we present a fundamental consideration of possible mechanisms for this oxidation reaction by using small molecular model catalysts, Raman spectra, static secondary ion mass spectroscopy (SIMS), DFT calculations, quasi in situ ATR-IR and UV spectra. Our study indicates that the defects, being favorable for the formation of active oxygen species, are the active sites for this oxidation reaction. Furthermore, one type of active defect, namely the armchair configuration defect was successfully identified.

  10. An experimental comparison of laboratory techniques in determining bulk properties of tuffaceous rocks; Yucca Mountain Site Characterization Project

    SciTech Connect

    Boyd, P.J.; Martin, R.J. III; Price, R.H.

    1994-04-01

    Samples of tuffaceous rock were studied as part of the site characterization for a potential nuclear waste repository at Yucca Mountain in southern Nevada. These efforts were scoping in nature, and their results, along with those of other investigations, are being used to develop suitable procedures for determining bulk properties of tuffaceous rock in support of thermal and mechanical properties evaluations. Comparisons were made between various sample preparation, handling, and measurement techniques for both zeolitized and nonzeolitized tuff in order to assess their effects on bulk property determinations. Laboratory tests included extensive drying regimes to evaluate dehydration behavior, the acquisition of data derived from both gas and water pycnometers to compare their suitability in determining grain densities, a comparison of particle size effects, and a set of experiments to evaluate whole core saturation methods. The results affirm the added complexity of these types of measurements where there is a zeolite component in the sample mineralogy. Absolute values for the bulk properties of zeolitized tuff are immeasurable due to the complex nature of their dehydration behavior. However, the results of the techniques that were investigated provide a basis for the development of preferred, consistent methods for determining the grain density, dry and saturated bulk densities, and porosity of tuffaceous rock, including zeolitic tuff in support of thermal and mechanical properties evaluations.

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

    PubMed Central

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

    2014-01-01

    Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S1P1 recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease. PMID:24667638

  12. 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

  13. Active Army and Army Reserve Soldiers: A Comparison.

    ERIC Educational Resources Information Center

    Corkan, JoJo T.; And Others

    A study determined whether chemical operations specialists at skill level 1 differ in terms of aptitude, job knowledge, job confidence, and perceptions of task difficulty, task importance, task frequency, and task training, depending on whether the specialists are active U.S. Army soldiers or are in the Army Reserve. The subjects for whom complete…

  14. 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

  15. Site specific comparison of H2, CH4 and compressed air energy storage in porous formations

    NASA Astrophysics Data System (ADS)

    Tilmann Pfeiffer, Wolf; Wang, Bo; Bauer, Sebastian

    2016-04-01

    The supply of energy from renewable sources like wind or solar power is subject to fluctuations determined by the climatic and weather conditions, and shortage periods can be expected on the order of days to weeks. Energy storage is thus required if renewable energy dominates the total energy production and has to compensate the shortages. Porous formations in the subsurface could provide large storage capacities for various energy carriers, such as hydrogen (H2), synthetic methane (CH4) or compressed air (CAES). All three energy storage options have similar requirements regarding the storage site characteristics and consequently compete for suitable subsurface structures. The aim of this work is to compare the individual storage methods for an individual storage site regarding the storage capacity as well as the achievable delivery rates. This objective is pursued using numerical simulation of the individual storage operations. In a first step, a synthetic anticline with a radius of 4 km, a drop of 900 m and a formation thickness of 20 m is used to compare the individual storage methods. The storage operations are carried out using -depending on the energy carrier- 5 to 13 wells placed in the top of the structure. A homogeneous parameter distribution is assumed with permeability, porosity and residual water saturation being 500 mD, 0.35 and 0.2, respectively. N2 is used as a cushion gas in the H2 storage simulations. In case of compressed air energy storage, a high discharge rate of 400 kg/s equating to 28.8 mio. m³/d at surface conditions is required to produce 320 MW of power. Using 13 wells the storage is capable of supplying the specified gas flow rate for a period of 31 hours. Two cases using 5 and 9 wells were simulated for both the H2 and the CH4 storage operation. The target withdrawal rates of 1 mio. sm³/d are maintained for the whole extraction period of one week in all simulations. However, the power output differs with the 5 well scenario producing

  16. 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

  17. Evidence for surface Ag + complexes as the SERS-active sites on Ag electrodes

    NASA Astrophysics Data System (ADS)

    Watanabe, T.; Kawanami, O.; Honda, K.; Pettinger, B.

    1983-12-01

    Evidence is given that SERS-active sites at Ag electrodes are associated with Ag + ions, forming sparingly soluble surface complexes with ligands such as pyridine molecules and halide ions. Such surface Ag + complexes contribute a factor of >800 to the overall (10 7-fold) enhancement, possibly via a resonance Raman effect.

  18. 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…

  19. 78 FR 18576 - Agency Information Collection Activities; Comment Request; Experimental Sites Data Collection...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-27

    ... Agency Information Collection Activities; Comment Request; Experimental Sites Data Collection Instrument... information collection requirements and provide the requested data in the desired format. ED is soliciting... collection instrument will be used to collect specific information/performance data for analysis of...

  20. Penicillin Use in Meningococcal Disease Management: Active Bacterial Core Surveillance Sites, 2009

    PubMed Central

    Blain, Amy E.; Mandal, Sema; Wu, Henry; MacNeil, Jessica R.; Harrison, Lee H.; Farley, Monica M.; Lynfield, Ruth; Miller, Lisa; Nichols, Megin; Petit, Sue; Reingold, Arthur; Schaffner, William; Thomas, Ann; Zansky, Shelley M.; Anderson, Raydel; Harcourt, Brian H.; Mayer, Leonard W.; Clark, Thomas A.; Cohn, Amanda C.

    2016-01-01

    In 2009, in the Active Bacterial Core surveillance sites, penicillin was not commonly used to treat meningococcal disease. This is likely because of inconsistent availability of antimicrobial susceptibility testing and ease of use of third-generation cephalosporins. Consideration of current practices may inform future meningococcal disease management guidelines. PMID:27704009

  1. 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…

  2. 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... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on the.... BOEM may issue one or more commercial wind energy leases in the WEA. The competitive lease process...

  3. 77 FR 3460 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-24

    ... published a final rule under 10 CFR Part 765 in the Federal Register on May 23, 1994, (59 FR 26714) to carry... for reimbursement. DOE amended the final rule on June 3, 2003, (68 FR 32955) to adopt several... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department...

  4. 75 FR 71677 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-24

    ... published a final rule under 10 CFR Part 765 in the Federal Register on May 23, 1994, (59 FR 26714) to carry... for reimbursement. DOE amended the final rule on June 3, 2003, (68 FR 32955) to adopt several... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department...

  5. 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.

  6. Laparoscopic ovariectomy in dogs: comparison between laparoendoscopic single-site and three-portal access

    PubMed Central

    Díaz-Güemes Martin-Portugués, Idoia; Fresno Bermejo, Laura; Sánchez-Margallo, Francisco Miguel

    2015-01-01

    This study was conducted to evaluate the feasibility and therapeutic safety of laparoendoscopic single-site ovariectomy (LESS-OVE) and 3-portal laparoscopic ovariectomy (Lap-OVE) in dogs. Ten female mixed breed dogs were included in the study. Dogs were divided into group 1 (LESS-OVE; n = 5) and group 2 (Lap-OVE; n = 5). All procedures were performed by laparoscopic-skilled surgeons, and the anesthetic protocol was the same for all patients. In both groups, the ovarian vascular pedicle and ligaments were transected using a bipolar vessel sealer/divider device. The mean total surgical time was slightly longer in LESS-OVE (36.6 ± 3.5 min) than Lap-OVE (32.0 ± 3.0 min); however, the differences were not significant. Perioperative complications were not reported in any group. Both laparoscopic techniques were shown to be equally feasible and safe for patients. However, surgeons found LESS-OVE to require more skill than Lap-OVE. Therefore, additional studies should be conducted to evaluate this novel approach in clinical veterinary practice, and a proper laparoscopic training program for veterinary surgeons should be developed. PMID:26119164

  7. Comparison of transfer sites for flexor digitorum longus in a cadaveric adult acquired flatfoot model.

    PubMed

    Vaudreuil, Nicholas J; Ledoux, William R; Roush, Grant C; Whittaker, Eric C; Sangeorzan, Bruce J

    2014-01-01

    Posterior tibialis tendon (PTT) dysfunction (PTTD) is associated with adult acquired flatfoot deformity. PTTD is commonly treated with a flexor digitorum longus (FDL) tendon transfer (FDLTT) to the navicular (NAV), medial cuneiform (CUN), or distal residuum of the degraded PTT (rPTT). We assessed the kinetic and kinematic outcomes of these three attachment sites using cadaveric gait simulation. Three transfer locations (NAV, CUN, rPTT) were tested on seven prepared flatfoot models using a robotic gait simulator (RGS). The FDLTT procedures were simulated by pulling on the PTT with biomechanically realistic FDL forces (rPTT) or by pulling on the transected FDL tendon after fixation to the navicular or medial cuneiform (NAV and CUN, respectively). Plantar pressure and foot bone motion were quantified. Peak plantar pressure significantly decreased from the flatfoot condition at the first metatarsal (NAV) and hallux (CUN). No difference was found in the medial-lateral center of pressure. Kinematic findings showed minimal differences between flatfoot and FDLTT specimens. The three locations demonstrated only minimal differences from the flatfoot condition, with the NAV and CUN procedures resulting in decreased medial pressures. Functionally, all three surgical procedures performed similarly.

  8. Comparison of hydraulic tomography with traditional methods at a highly heterogeneous site.

    PubMed

    Berg, Steven J; Illman, Walter A

    2015-01-01

    Over the past several decades, different groundwater modeling approaches of various complexities and data use have been developed. A recently developed approach for mapping hydraulic conductivity (K) and specific storage (Ss ) heterogeneity is hydraulic tomography, the performance of which has not been compared to other more "traditional" methods that have been utilized over the past several decades. In this study, we compare seven methods of modeling heterogeneity which are (1) kriging, (2) effective parameter models, (3) transition probability/Markov Chain geostatistics models, (4) geological models, (5) stochastic inverse models conditioned to local K data, (6) hydraulic tomography, and (7) hydraulic tomography conditioned to local K data using data collected in five boreholes at a field site on the University of Waterloo (UW) campus, in Waterloo, Ontario, Canada. The performance of each heterogeneity model is first assessed during model calibration. In particular, the correspondence between simulated and observed drawdowns is assessed using the mean absolute error norm, (L1 ), mean square error norm (L2 ), and correlation coefficient (R) as well as through scatterplots. We also assess the various models on their ability to predict drawdown data not used in the calibration effort from nine pumping tests. Results reveal that hydraulic tomography is best able to reproduce these tests in terms of the smallest discrepancy and highest correlation between simulated and observed drawdowns. However, conditioning of hydraulic tomography results with permeameter K data caused a slight deterioration in accuracy of drawdown predictions which suggests that data integration may need to be conducted carefully.

  9. Comparison of Vaisala radiosondes RS41 and RS92 at the ARM Southern Great Plains Site

    DOE PAGES

    Jensen, M. P.; Holdridge, D.; Survo, P.; Lehtinen, R.; Baxter, S.; Toto, T.; Johnson, K. L.

    2015-11-02

    In the fall of 2013, the Vaisala RS41-SG (4th generation) radiosonde was introduced as a replacement for the RS92-SGP radiosonde with improvements in measurement accuracy of profiles of atmospheric temperature, humidity and pressure. Thus, in order to help characterize these improvements, an intercomparison campaign was undertaken at the US Department of Energy's Atmospheric Radiation Measurement (ARM) Facility site in north Central Oklahoma USA. During 3–8 June 2014, a total of 20 twin-radiosonde flights were performed in a variety of atmospheric conditions representing typical midlatitude continental summertime conditions. The results suggest that the RS92 and RS41 measurements generally agree within manufacturermore » specified tolerances with notable exceptions when exiting liquid cloud layers where the "wet bulbing" effect is mitigated in the RS41 observations. The RS41 measurements also appear to show a smaller impact from solar heating. These results suggest that the RS41 does provide important improvements, particularly in cloudy conditions, but under most observational conditions the RS41 and RS92 measurements agree within the manufacturer specified limits and so a switch to RS41 radiosondes will have little impact on long-term observational records.« less

  10. Comparison of transfer sites for flexor digitorum longus in a cadaveric adult acquired flatfoot model.

    PubMed

    Vaudreuil, Nicholas J; Ledoux, William R; Roush, Grant C; Whittaker, Eric C; Sangeorzan, Bruce J

    2014-01-01

    Posterior tibialis tendon (PTT) dysfunction (PTTD) is associated with adult acquired flatfoot deformity. PTTD is commonly treated with a flexor digitorum longus (FDL) tendon transfer (FDLTT) to the navicular (NAV), medial cuneiform (CUN), or distal residuum of the degraded PTT (rPTT). We assessed the kinetic and kinematic outcomes of these three attachment sites using cadaveric gait simulation. Three transfer locations (NAV, CUN, rPTT) were tested on seven prepared flatfoot models using a robotic gait simulator (RGS). The FDLTT procedures were simulated by pulling on the PTT with biomechanically realistic FDL forces (rPTT) or by pulling on the transected FDL tendon after fixation to the navicular or medial cuneiform (NAV and CUN, respectively). Plantar pressure and foot bone motion were quantified. Peak plantar pressure significantly decreased from the flatfoot condition at the first metatarsal (NAV) and hallux (CUN). No difference was found in the medial-lateral center of pressure. Kinematic findings showed minimal differences between flatfoot and FDLTT specimens. The three locations demonstrated only minimal differences from the flatfoot condition, with the NAV and CUN procedures resulting in decreased medial pressures. Functionally, all three surgical procedures performed similarly. PMID:24115238

  11. Spontaneous vegetation succession at different central European mining sites: a comparison across seres.

    PubMed

    Prach, Karel; Lencová, Kamila; Rehounková, Klára; Dvořáková, Helena; Jírová, Alena; Konvalinková, Petra; Mudrák, Ondřej; Novák, Jan; Trnková, Romana

    2013-11-01

    We performed detrended correspondence analysis (DCA) ordination to compare seven successional seres running in stone quarries, coal mining spoil heaps, sand and gravel pits, and extracted peatlands in the Czech Republic in central Europe. In total, we obtained 1,187 vegetation samples containing 705 species. These represent various successional stages aged from 1 to 100 years. The successional seres studied were more similar in their species composition in the initial stages, in which synathropic species prevailed, than in later successional stages. This vegetation differentiation was determined especially by local moisture conditions. In most cases, succession led to a woodland, which usually established after approximately 20 years. In very dry or wet places, by contrast, where woody species were limited, often highly valuable, open vegetation developed. Except in the peatlands, the total number of species and the number of target species increased during succession. Participation of invasive aliens was mostly unimportant. Spontaneous vegetation succession generally appears to be an ecologically suitable and cheap way of ecosystem restoration of heavily disturbed sites. It should, therefore, be preferred over technical reclamation. PMID:23436061

  12. Comparison of the response to endovascular reperfusion in relation to site of arterial occlusion

    PubMed Central

    Mlynash, Michael; Straka, Matus; Kemp, Stephanie; Bammer, Roland; Marks, Michael P.; Albers, Gregory W.; Lansberg, Maarten G.

    2013-01-01

    Objective: We explored the relationship between the site of vascular occlusion and the response to endovascular treatment in patients with acute ischemic stroke and also considered the impact of mismatch profile. Methods: DEFUSE-2 was a prospective cohort study of patients treated with endovascular therapy. Patients with internal carotid artery (ICA) and middle cerebral artery (MCA) involvement were included in this substudy. Mismatch and reperfusion status was assessed on MRI. Favorable clinical response was defined as an improvement of at least 8 points on the NIH Stroke Scale. Results: Reperfusion rates were comparable in both groups (61% for ICA and 59% for MCA). In the setting of reperfusion, percentages of favorable clinical response were similar between patients with stroke due to ICA (65%) and MCA (63%) occlusions. When reperfusion was not achieved, favorable outcomes were less frequent with obstructions of the ICA (9%) than the MCA (52%). Among target mismatch patients, the adjusted odds ratio for favorable clinical response associated with reperfusion was 39.7 (95% confidence interval 1.4–1,132.8) for ICA occlusions vs 5.1 (95% confidence interval 1.4–19.3) for MCA occlusions. Conclusions: Endovascular reperfusion is associated with favorable clinical response regardless of the location of the arterial occlusion. This association is strongest for target mismatch patients with ICA occlusions. Target mismatch patients with either ICA or MCA occlusions appear to be good candidates for endovascular reperfusion therapy. PMID:23851962

  13. Comparison of Vaisala radiosondes RS41 and RS92 at the ARM Southern Great Plains site

    NASA Astrophysics Data System (ADS)

    Jensen, Michael P.; Holdridge, Donna J.; Survo, Petteri; Lehtinen, Raisa; Baxter, Shannon; Toto, Tami; Johnson, Karen L.

    2016-07-01

    In the fall of 2013, the Vaisala RS41 (fourth generation) radiosonde was introduced as a replacement for the RS92-SGP radiosonde with improvements in measurement accuracy of profiles of atmospheric temperature, humidity, and pressure. In order to help characterize these improvements, an intercomparison campaign was undertaken at the US Department of Energy's Atmospheric Radiation Measurement (ARM) Climate Research Facility site in north-central Oklahoma, USA. During 3-8 June 2014, a total of 20 twin-radiosonde flights were performed in a variety of atmospheric conditions representing typical midlatitude continental summertime conditions. The results show that for most of the observed conditions the RS92 and RS41 measurements agree much better than the manufacturer-specified combined uncertainties with notable exceptions when exiting liquid cloud layers where the "wet-bulbing" effect appears to be mitigated for several cases in the RS41 observations. The RS41 measurements of temperature and humidity, with applied correction algorithms, also appear to show less sensitivity to solar heating. These results suggest that the RS41 does provide important improvements, particularly in cloudy conditions. For many science applications - such as atmospheric process studies, retrieval development, and weather forecasting and climate modeling - the differences between the RS92 and RS41 measurements should have little impact. However, for long-term trend analysis and other climate applications, additional characterization of the RS41 measurements and their relation to the long-term observational records will be required.

  14. Comparison of Vaisala radiosondes RS41 and RS92 at the ARM Southern Great Plains Site

    SciTech Connect

    Jensen, M. P.; Holdridge, D.; Survo, P.; Lehtinen, R.; Baxter, S.; Toto, T.; Johnson, K. L.

    2015-11-02

    In the fall of 2013, the Vaisala RS41-SG (4th generation) radiosonde was introduced as a replacement for the RS92-SGP radiosonde with improvements in measurement accuracy of profiles of atmospheric temperature, humidity and pressure. Thus, in order to help characterize these improvements, an intercomparison campaign was undertaken at the US Department of Energy's Atmospheric Radiation Measurement (ARM) Facility site in north Central Oklahoma USA. During 3–8 June 2014, a total of 20 twin-radiosonde flights were performed in a variety of atmospheric conditions representing typical midlatitude continental summertime conditions. The results suggest that the RS92 and RS41 measurements generally agree within manufacturer specified tolerances with notable exceptions when exiting liquid cloud layers where the "wet bulbing" effect is mitigated in the RS41 observations. The RS41 measurements also appear to show a smaller impact from solar heating. These results suggest that the RS41 does provide important improvements, particularly in cloudy conditions, but under most observational conditions the RS41 and RS92 measurements agree within the manufacturer specified limits and so a switch to RS41 radiosondes will have little impact on long-term observational records.

  15. Comparison of antifungal activities of Vietnamese citrus essential oils.

    PubMed

    Van Hung, Pham; Chi, Pham Thi Lan; Phi, Nguyen Thi Lan

    2013-03-01

    Citrus essential oils (EOs) are volatile compounds from citrus peels and widely used in perfumes, cosmetics, soaps and aromatherapy. In this study, inhibition of citrus EOs extracted from Vietnamese orange (Citrus sinensis), mandarin (Citrus reticulata Blanco), pomelo (Citrus grandis Osbeck) and lime (Citrus aurantifolia Swingle) on the growth of plant pathogenic fungi, Mucor hiemalis, Penicillium expansum and Fusarium proliferatum was investigated. The EOs of the citrus peels were obtained by cold-pressing method and the antifungal activity of EOs was evaluated using the agar dilution method. The results show that the EOs had significant antifungal activity. Lime EO was the best inhibitor of M. hiemalis and F. proliferatum while pomelo EO was the most effective against P. expansum. These results indicate that citrus EOs can be used as antifungal natural products in the food, pharmaceutical and cosmetic industries.

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

    PubMed

    Taylor, John C; Markham, George D

    2003-07-15

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

  17. Comparison Of NaI And HPGe Minimum Detectable Activities

    SciTech Connect

    Bailey, Paul

    2002-11-30

    The Minimum Detectable Activity of a 76 mm by 76 mm (3" by 3") sodium iodide (NaI) crystal and 18 %, 42 % and 68 % efficient HPGe detectors were calculated and compared for gamma-ray spectrometry with count times in the range of 1 second to 15 minutes. All cases were for in situ measurements with a surface distribution source and a detector height of 1 meter. The radionuclides considered were 137Cs and 60Co.

  18. An Inter-Comparison of Two Independent Site Test Interferometers Located in Goldstone, California: Initial Study Results

    NASA Technical Reports Server (NTRS)

    Morabito, David; D'Addario, Larry; Acosta, Roberto J.; Nessel, James A.

    2012-01-01

    Site Test Interferometers (STIs) have been deployed at two different locations at the NASA Deep Space Network (DSN) tracking complex in Goldstone, California. An STI measures the difference in path length between a geostationary satellite and two antennas on the Earth, producing a measure of atmospheric phase fluctuations over spatial distances comparable to those between elements of possible microwave phased arrays. The purposes of the Goldstone STIs are to assess the suitability of Goldstone as an array site and to statistically characterize atmospheric induced delay fluctuations for application to future array scenarios.The two STI's are separated by 13 km across the Goldstone complex. Each instrument is composed of two small-diameter antennas and associated electronics separated by approx. 200 meters in a principally east-west configuration. The antennas continuously observe signals emitted by geo-stationary satellites and produce data that contain information on the phase difference between signals received by both antennas. The fluctuation in delay (or path length difference) statistics derived from these data sets can be used to infer power loss for particular array configurations.We report on a comparison of the root mean square (RMS) phase delay statistics estimated over 10-minute intervals between the two Goldstone STIs. We have achieved good statistical agreement between the data acquired from the two STIs, given that each instrument is observing different satellites, at different frequencies, over different baseline lengths, with very different implementations, and are located 13 km apart in widely separated terrain at different geodetic altitudes.

  19. A comparison of cloud properties at a coastal and inland site at the North Slope of Alaska

    DOE PAGES

    Doran, J. C.; Zhong, S.; Liljegren, J. C.; Jakob, C.

    2002-06-11

    In this study, we have examined differences in cloud liquid water paths (LWPs) at a coastal (Barrow) and an inland (Atqasuk) location on the North Slope of Alaska using microwave radiometer (MWR) data collected by the U.S. Department of Energy's Atmospheric Radiation Measurement program for the period June-September 1999. Revised retrieval procedures and a filtering algorithm to eliminate data contaminated by wet windows on the MWRs were employed to extract high-quality data suitable for this study. For clouds with low base heights (<350 m), the LWPs at the coastal site were significantly higher than those at the inland site, butmore » for clouds with higher base heights the differences were small. Air-surface interactions may account for some of the differences. Comparisons were also made between observed LWPs and those simulated with the European Centre for Medium-Range Weather Forecasts (ECMWF) model. The model usually successfully captured the occurrence of cloudy periods but it underpredicted the LWPs by approximately a factor of two. It was also unsuccessful in reproducing the observed differences in LWPs between Barrow and Atqasuk. Some suggestions on possible improvements in the model are presented.« less

  20. A comparison of cloud properties at a coastal and inland site at the North Slope of Alaska

    SciTech Connect

    Doran, J. C.; Zhong, S.; Liljegren, J. C.; Jakob, C.

    2002-06-11

    In this study, we have examined differences in cloud liquid water paths (LWPs) at a coastal (Barrow) and an inland (Atqasuk) location on the North Slope of Alaska using microwave radiometer (MWR) data collected by the U.S. Department of Energy's Atmospheric Radiation Measurement program for the period June-September 1999. Revised retrieval procedures and a filtering algorithm to eliminate data contaminated by wet windows on the MWRs were employed to extract high-quality data suitable for this study. For clouds with low base heights (<350 m), the LWPs at the coastal site were significantly higher than those at the inland site, but for clouds with higher base heights the differences were small. Air-surface interactions may account for some of the differences. Comparisons were also made between observed LWPs and those simulated with the European Centre for Medium-Range Weather Forecasts (ECMWF) model. The model usually successfully captured the occurrence of cloudy periods but it underpredicted the LWPs by approximately a factor of two. It was also unsuccessful in reproducing the observed differences in LWPs between Barrow and Atqasuk. Some suggestions on possible improvements in the model are presented.

  1. Anti-inflammatory activity of Bromelia hieronymi: comparison with bromelain.

    PubMed

    Errasti, María E; Caffini, Néstor O; Pelzer, Lilian E; Rotelli, Alejandra E

    2013-03-01

    Some plant proteases (e. g., papain, bromelain, ficin) have been used as anti-inflammatory agents for some years, and especially bromelain is still being used as alternative and/or complementary therapy to glucocorticoids, nonsteroidal antirheumatics, and immunomodulators. Bromelain is an extract rich in cysteine endopeptidases obtained from Ananas comosus. In this study the anti-inflammatory action of a partially purified extract of Bromelia hieronymi fruits, whose main components are cysteine endopeptidases, is presented. Different doses of a partially purified extract of B. hieronymi were assayed on carrageenan-induced and serotonine-induced rat paw edema, as well as in cotton pellet granuloma model. Doses with equal proteolytic activity of the partially purified extract and bromelain showed significantly similar anti-inflammatory responses. Treatment of the partially purified extract and bromelain with E-64 provoked loss of anti-inflammatory activity on carrageenan-induced paw edema, a fact which is consistent with the hypothesis that the proteolytic activity would be responsible for the anti-inflammatory action.

  2. Spectroscopic studies of single and double variants of M ferritin: lack of conversion of a biferrous substrate site into a cofactor site for O2 activation.

    PubMed

    Kwak, Yeonju; Schwartz, Jennifer K; Haldar, Suranjana; Behera, Rabindra K; Tosha, Takehiko; Theil, Elizabeth C; Solomon, Edward I

    2014-01-28

    Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O2 to react with substrate. Ferritin has an active site ligand set with 1-His/4-carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O2 or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O2 activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a μ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O2 reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O2 by the binuclear non-heme iron enzymes.

  3. Characterization of the active site properties of CYP4F12.

    PubMed

    Eksterowicz, John; Rock, Dan A; Rock, Brooke M; Wienkers, Larry C; Foti, Robert S

    2014-10-01

    Cytochrome P450 4F12 is a drug-metabolizing enzyme that is primarily expressed in the liver, kidney, colon, small intestine, and heart. The properties of CYP4F12 that may impart an increased catalytic selectivity (decreased promiscuity) were explored through in vitro metabolite elucidation, kinetic isotope effect experiments, and computational modeling of the CYP4F12 active site. By using astemizole as a probe substrate for CYP4F12 and CYP3A4, it was observed that although CYP4F12 favored astemizole O-demethylation as the primary route of metabolism, CYP3A4 was capable of metabolizing astemizole at multiple sites on the molecule. Deuteration of astemizole at the site of O-demethylation resulted in an isotope effect of 7.1 as well as an 8.3-fold decrease in the rate of clearance for astemizole by CYP4F12. Conversely, although an isotope effect of 3.8 was observed for the formation of the O-desmethyl metabolite when deuterated astemizole was metabolized by CYP3A4, there was no decrease in the clearance of astemizole. Development of a homology model of CYP4F12 based on the crystal structure of cytochrome P450 BM3 predicted an active site volume for CYP4F12 that was approximately 76% of the active site volume of CYP3A4. As predicted, multiple favorable binding orientations were available for astemizole docked into the active site of CYP3A4, but only a single binding orientation with the site of O-demethylation oriented toward the heme was identified for CYP4F12. Overall, it appears that although CYP4F12 may be capable of binding similar ligands to other cytochrome P450 enzymes such as CYP3A4, the ability to achieve catalytically favorable orientations may be inherently more difficult because of the increased steric constraints of the CYP4F12 active site. PMID:25074871

  4. A modular treatment of molecular traffic through the active site of cholinesterase

    PubMed Central

    Botti, SA; Felder, CE; Lifson, S; Sussman, JL; Silman, I

    1999-01-01

    We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products. PMID:10545346

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

    SciTech Connect

    Yip, Wing-Kin; Dong, Jian-Guo; Yang, S.F. ); Kenny, J.W.; Thompson, G.A. )

    1990-10-01

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

  6. Retroperitoneal Laparoendoscopic Single-Site Ureterolithotomy: A Comparison with Conventional Laparoscopic Surgery

    PubMed Central

    Liu, Xiaopeng; Huang, Huaiqiu; Wu, Jieying; Huang, Wentao; Cai, Songwang; Li, Xiaojuan; Ye, Chunwei; Zhu, Baoyi; Cai, Yi; Gao, Xin

    2012-01-01

    Abstract Background and Purpose Laparoendoscopic single-site (LESS) surgery through the retroperitoneal approach has been seldom reported. We aimed to compare the feasibility and outcomes of LESS and conventional laparoscopic surgery via the retroperitoneal approach in the management of large, impacted ureteral stones. Patients and Methods From June 2010 to May 2011, LESS ureterolithotomy through the retroperitoneal approach was performed in 10 patients (the LESS group). Another 15 patients who underwent conventional retroperitoneal laparoscopic ureterolithotomy (the conventional laparoscopic group) by the same surgeon were involved and compared. The operative time, complications, and surgical outcomes were evaluated. Results All the operations were completed successfully, without conversion to conventional laparoscopic or open surgeries. The operative time of the LESS group and of the conventional laparoscopic group were 132.7±16.3 and 128.1±20.1 minutes, respectively (P=0.782). The estimated blood loss were 30.7±5.9 vs 28.0±4.5 mL (P=0.620). Duration of analgesia postoperatively was 2.0±0.8 vs 3.5±0.5 days (P=0.005). All targeted stones were successfully extracted without major complications. Postoperative urine leakage was noted in one patient in each group. Cosmetic results were superior in the LESS group according to both the study nurse's and the patients' assessments (8.5 vs 5.3; P=0.012, and 8.3 vs 5.6; P=0.025, respectively). All patients showed no obstructions or stricture formations on postoperative follow-up. Conclusions In experienced hands, LESS for ureterolithotomy through the retroperitoneal approach is feasible and can acquire outcomes equal to those of conventional multiport laparoscopic surgery. Prospective long-term follow-up studies with a larger number of patients are needed to further evaluate its benefits. PMID:22103789

  7. Laparoendoscopic Single-Site Pyeloplasty Using Additional 2 mm Instruments: A Comparison with Conventional Laparoscopic Pyeloplasty

    PubMed Central

    Ju, Sung Ho; Lee, Dong-Gi; Lee, Jun Ho; Baek, Min Ki; Jeong, Byong Chang; Jeon, Seong Soo; Lee, Kyu-Sung

    2011-01-01

    Purpose Despite a recent surge in the performance of laparoendoscopic single-site surgery (LESS), concerns remain about performing LESS pyeloplasty (LESS-P) because of the technical difficulty in suturing. We report our techniques and initial experiences with LESS-P using additional needlescopic instruments and compare the results with conventional laparoscopic pyeloplasty (CL-P). Materials and Methods Nine patients undergoing LESS-P were matched 2:1 with regard to age and side of surgery to a previous cohort of 18 patients who underwent CL-P. In both groups, the operating procedures were performed equally except for the number of access points. In the LESS-P group, we made a single 2 cm incision at the umbilicus and used a homemade port. We also used additional 2 mm needlescopic instruments at the subcostal area to facilitate suturing and the ureteral stenting. Results The preoperative characteristics were comparable in both groups. Postoperatively, no significant differences were noted between the LESS-P and CL-P cases in regard to length of stay, estimated blood loss, analgesics required, and complications. But, LESS-P was associated with a shorter operative time (252.2 vs. 309.7 minutes, p=0.044) and less pain on postoperative day one (numeric rating scale 3.7 vs. 5.6, p=0.024). The success rate was 94% with CL-P (median, 23 months) and 100% with LESS-P (median, 14 months). Conclusions Our initial experiences suggest that LESS-P is a feasible and safe procedure. The use of additional 2 mm instruments can help to overcome the difficulties associated with LESS surgery. PMID:22025957

  8. Solar Radiation Measurements at the Chesapeake Bay COVE Site and Comparison With Model

    NASA Astrophysics Data System (ADS)

    Jin, Z.; Charlock, T.; Rutledge, K.

    2001-05-01

    To validate retrievals of flux and albedo in the CERES satellite program, broad-band upwelling and downwelling solar irradiances are measured routinely at the CERES Ocean Validation Experiment (COVE) site 25 km east of the coast of Virginia, near the mouth of the Chesapeake Bay. A full year of observations are compared with simulations from a coupled radiative transfer model. The coupled model treats absorption and scattering by layers of both the atmosphere and the ocean explicitly and consistently, in terms of the inherent optical properties of the air and the sea. Key input parameters for the model include aerosol optical depth, wind speed, and total precipitable water; these are measured at COVE. The modeled total downwelling irradiances, which depend mainly on the atmospheric optical properties, agree well with observations. But the modeled upwelling irradiances (and hence ocean surface albedo), which depend heavily on the the ocean optical properties, are generally less than the observations. The measured upwelling irradiances are strongly influenced by sea state and surface wind, resulting in a seasonal variation of the ocean surface albedo. Candidates to explain the discrepancy of observed and modeled albedo are (1) in-ocean scattering that was not included in the model (i.e., sediments or air bubbles), (2) possible inadequacy of the classical Cox-Munk distribution for the wind speed dependence of sea slopes, and (3) uncertainties in aerosol optical properties. We are presently testing SeaWiFS data as a source for the concentrations of chlorophyl and dissolved organic matter (DOM); and plan to compare the model with available upwelling spectral irradiances and radiances, in addition to the broadband fluxes as described above.

  9. COMPARISON OF THREE TRACER TESTS AT THE RAFT RIVER GEOTHERMAL SITE

    SciTech Connect

    Earl D Mattson; Mitchell Plummer; Carl Palmer; Larry Hull; Samantha Miller; Randy Nye

    2011-02-01

    Three conservative tracer tests have been conducted through the Bridge Fault fracture zone at the Raft River Geothermal (RRG) site. All three tests were conducted between injection well RRG-5 and production wells RRG-1 (790 m distance) and RRG-4 (740 m distance). The injection well is used during the summer months to provide pressure support to the production wells. The first test was conducted in 2008 using 136 kg of fluorescein tracer. Two additional tracers were injected in 2010. The first 2010 tracer injected was 100 kg fluorescein disodium hydrate salt on June, 21. The second tracer (100 kg 2,6-naphthalene disulfonic acid sodium salt) was injected one month later on July 21. Sampling of the two productions wells is still being performed to obtain the tail end of the second 2010 tracer test. Tracer concentrations were measured using HPLC with a fluorescence detector. Results for the 2008 test, suggest 80% tracer recover at the two production wells. Of the tracer recovered, 85% of tracer mass was recovered in well RRG-4 indicating a greater flow pathway connection between injection well and RRG-4 than RRG-1. Fluorescein tracer results appear to be similar between the 2008 and 2010 tests for well RRG-4 with peak concentrations arriving approximately 20 days after injection despite the differences between the injection rates for the two tests (~950 gpm to 475 gpm) between the 2008 and 2010. The two 2010 tracer tests will be compared to determine if the results support the hypothesis that rock contraction along the flow pathway due to the 55 oC cooler water injection alters the flow through the ~140 oC reservoir.

  10. Immediate loaded dental implants: comparison between fixtures inserted in postextractive and healed bone sites.

    PubMed

    Degidi, Marco; Piattelli, Adriano; Carinci, Francesco

    2007-07-01

    In the last two decades, several investigators have reported immediate placement of dental implants into extraction sockets achieving excellent results with a two-stage surgical procedure. Recently, immediate loading (IL) has become an emerging technique because it has been documented to be a successful and time-saving procedure. Regarding the possibility of immediate/early loading of implants placed in fresh extraction sockets, few reports are available. In addition, they are based on limited series with short follow up. Thus, we decided to perform a retrospective study on a large series of postextractive IL implants. From January 1995 to October 2004, 416 IL fixtures were placed immediately after extraction and 658 IL fixtures in healed sites. The mean follow up is 3 years. Multiple implant systems were used. Because only eight of 1,074 implants were lost (i.e., survival rate, 99.3%) and no statistical differences were detected among the studied variables, no or reduced marginal bone loss was considered as an indicator of success rate to evaluate the effect of several host-, surgery-, and implant-related factors. A general linear model was then performed to detect those variables statistically associated with marginal bone loss. Only eight of 1,074 implants were lost (i.e., survival rate, 99.3%) and no differences were detected among the studied variables. On the contrary, the general linear model showed that younger age (cutoff, 55 years) and harder bone are related to a lower delta insertion abutment junction (or marginal bone loss) and thus a better outcome. We demonstrated that postextractive IL implants have a high survival rate and success rate that are similar to those reported in previous studies of two-stage procedures or in IL implants inserted in healed bone. Poor bone quality and older age correlate with a slight higher bone resorption.

  11. Comparison of stress-measuring techniques at the DNA-UTP site, Rodgers Hollow, Kentucky

    SciTech Connect

    Finley, R.E.

    1994-12-01

    The Defense Nuclear Agency (DNA) is developing explosives technology through its Underground Technology Program (UTP). Sandia National Laboratories (SNL) has supported the DNA by conducting research to characterize the in situ stress and rock mass deformability at one of the UTP underground sites at Rodgers Hollow, near Louisville, Kentucky on the Fort Knox Military Reservation. The purpose of SNL`s testing was to determine the in situ stress using three different measurement techniques and, if possible, to estimate the rock mass modulus near the underground opening. The three stress-measuring techniques are (1) borehole deformation measurements using overcoring, (2) Anelastic Strain Recovery (ASR) complemented by laboratory ultrasonic and mechanical properties testing, and (3) the in situ flatjack technique using cancellation pressure. Rock mass modulus around the underground opening was estimated using the load deformation history of the flatjack and surrounding rock. Borehole deformation measurements using the overcoring technique probably represent the most reliable method for in situ stress determination in boreholes up to 50 ft (15 m) deep in competent rock around an isolated excavation. The technique is used extensively by the tunneling and mining industries. The ASR technique is also a core-based technique and is used in the petroleum and natural gas industries for characterization of in situ stress from deep boreholes. The flatjack technique has also been used in the tunneling and mining industries, and until recently has been limited to measurement of the stress immediately around the excavation. Results from the flatjack technique must be further analyzed to calculate the in situ stress in the far field.

  12. Comparison of biogeochemical dynamics in two time-series sites of a North Atlantic Ocean site (PAP and BATS): a modeling approach.

    NASA Astrophysics Data System (ADS)

    Ibello, Valeria; Butenschon, Momme; Salihoglu, Baris; Erkan Kideys, Ahmet

    2013-04-01

    Plankton functional type (PFT) models are highly complex ecosystem models. Indeed, the large number of processes and plankton functional groups represented in these models make the network of interactions extremely complicated. Slight differences in parameterization or formulation of single processes, therefore, may drive these models to respond in a very different way to perturbations of the system. An evaluation of such a different responses can be very useful to understand the processes regulating the functioning of the ecosystem. In this study we analyze the sensitivity of the biological parameters in a PFT model (European Regional Seas Ecosystem Model, ERSEM) in respect to primary production and detrital export. The tests are done on a subset of key parameters that control ocean ecosystem growth in a 1-D formulation of ERSEM coupled with a turbulence model (General Ocean Turbulence Model, GOTM). Results are compared with observed data from two time-series sites Bermuda Atlantic Time-Series (BATS, 32.16 N 64.5 W) and Porcupine Abyssal Plain (PAP, 49 N 16 W). A particular focus on factors determining the timing and intensity of the bloom is also presented on the base of literature review and on 1D(GOTM-ERSEM)-3D(NEMO-ERSEM) model simulations comparison. The different processes evaluated are: i). winter convective mixing, ii) lateral advection: mesoscale and sub-mesoscale eddies, iii) turbolent mixing iv) decoupling between euphotic zone and mixed layer depth. The study presented here is carried out in the framework of the European project EURO-BASIN (European Basin-scale Analysis, Synthesis and Integration), where long term 3D simulation aimed to evaluate the variability of primary production and carbon export are planned. Parameterization in use by the 3D NEMO-ERSEM is referring to the global ocean, while simulations are planned for the North North Atlantic. This study aims to contribute to fulfill the development of a specific parameterization for the North

  13. Hydrogen bonding and spin density distribution in the Qb semiquinone of bacterial reaction centers and comparison with the Qa site.

    PubMed

    Martin, Erik; Samoilova, Rimma I; Narasimhulu, Kupala V; Lin, Tzu-Jen; O'Malley, Patrick J; Wraight, Colin A; Dikanov, Sergei A

    2011-04-13

    (B) function and comparisons with the Q(A) site are discussed.

  14. Lymphokine-activated killer (LAK) cells can be focused at sites of tumor growth by products of macrophage activation

    SciTech Connect

    Migliori, R.J.; Gruber, S.A.; Sawyer, M.D.; Hoffman, R.; Ochoa, A.; Bach, F.H.; Simmons, R.L.

    1987-08-01

    Successful adoptive cancer immunotherapy presumably depends on the accumulation of tumoricidal leukocytes at the sites of tumor growth. Large numbers of lymphokine-activated killer (LAK) cells can be generated in vitro by growth in high concentrations of interleukin-2 (IL-2), but relatively few arrive at the tumor site after intravenous injection. We hypothesize that the delivery of LAK cells to tumor sites may be augmented by previously demonstrated lymphocyte-recruiting factors, including activated macrophage products such as interleukin-1 (IL-1) and tumor necrosis factor. /sup 111/Indium-labeled LAK cells were injected intravenously into syngeneic mice bearing the macrophage activator endotoxin (LPS) in one hind footpad, and saline solution was injected into the contralateral footpad. Significantly more activity was recovered from the LPS-bearing footpad at all times during a 96-hour period. Recombinant IL-1 also attracted more LAK cells after injection into tumor-free hind footpads. Furthermore, LAK cells preferentially homed to hind footpads that were bearing 3-day established sarcomas after intralesional injections of LPS, IL-1, or tumor necrosis factor when compared with contralateral tumor-bearing footpads injected with saline solution alone. In preliminary experiments, mice with hind-footpad tumors appeared to survive longer after combined systemic IL-2 and LAK therapy if intralesional LPS was administered. These studies demonstrate that macrophage activation factors that have been shown capable of attracting circulating normal lymphocytes can also effectively attract LAK cells from the circulation. By the stimulation of macrophages at the sites of tumor growth, more LAK cells can be attracted. It is hoped that by focusing the migration of LAK cells to tumors, LAK cells and IL-2 would effect tumor regression more efficiently and with less toxicity.

  15. Comparison of solar activity during last two minima on turn of Activity Cycles 22/23 and 23/24

    NASA Astrophysics Data System (ADS)

    Gryciuk, Magdalena; Gburek, Szymon; Siarkowski, Marek; Podgorski, Piotr; Sylwester, Janusz; Farnik, Frantisek

    2013-07-01

    The subject of our work is the review and comparison of solar activity during the last two solar minima that occurred between recent activity cycles. We use the soft X-ray global solar corona observations covering the two nine-months long time intervals in 1997/98 and 2009. Data from RF15-I multichannel photometer are used for the penultimate minimum. For the last unusually deep and prolonged solar activity minimum in 2009 the data from SphinX spectrophotometer are used. Comparison of measurements from both minima takes place in the overlapping energy range 2-15 keV. We focus on the active region formation, evolution and flaring productivity during respective minima.

  16. Comparison of mussels and semi-permeable membrane devices as intertidal monitors of polycyclic aromatic hydrocarbons at oil spill sites.

    PubMed

    Boehm, Paul D; Page, David S; Brown, John S; Neff, Jerry M; Edward Bence, A

    2005-07-01

    Side-by-side comparisons of polycyclic aromatic hydrocarbon (PAH) concentrations in resident blue mussels (Mytilus trossulus) and in semi-permeable membrane devices (SPMDs) were made at four sites in Prince William Sound, Alaska. SPMDs were deployed for approximately 30 days on the surface of the beach sediment at three tidal elevations on each shore and in 0.5 m deep open pits in the middle intertidal zone. Total PAH (TPAH) concentrations in mussels and in SPMDs were correlated, but the PAH compositions were different. The lower molecular weight PAH were relatively more abundant in the SPMDs than in the mussels at oiled and HA sites. TPAH concentrations in SPMDs deployed in pits and mussels collected adjacent to those pits at oiled sites were higher than in SPMDs and mussels from non-pitted SPMD locations approximately 3-15 m from the pits. Pitting released buried oil making its PAH bioavailable. SPMDs deployed in the supratidal zone (+4.0 m tidal elevation) were exposed to atmospheric contaminants for a large fraction of the deployment time and accumulated primarily pyrogenic (combustion-sourced) PAH from the atmosphere. The SPMD strips supplied by the manufacturer contained significant amounts (approximately 125 ng/strip) of primarily alkylated 2-3 ring PAH. These blank levels make SPMDs unsuitable for shoreline assessments when environmental PAH concentrations are low. Consequently, where available, mussels are recommended for use in assessments of the bioavailability of buried oil residues sequestered in intertidal sediments following an oil spill. Mussels are the preferred monitoring tool when the assessments involve food-chain effects. At locations where the absence of mussels necessitates the use of SPMDs or other passive sampling devices, their limitations need to be carefully considered in the interpretation of results.

  17. Comparison of the CALIPSO satellite and ground-based observations of cirrus clouds at the ARM TWP sites

    SciTech Connect

    Thorsen, Tyler J.; Fu, Q.; Comstock, Jennifer M.

    2011-11-10

    Statistics of ice cloud macrophysical and optical properties from the Cloud-Aerosol LIdar with Orthogonal Polarization (CALIOP) instrument on board the Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observations (CALIPSO) satellite are compared with those from ground-based lidar observations over a 31 month period. Ground-based lidar observations are taken from the micropulse lidars (MPL) at the three Department of Energy Atmospheric Radiation Measurement (ARM) tropical western pacific (TWP) sites: Manus, Nauru and Darwin. CALIPSO observations show a larger cloud fraction at high altitudes while the ground-based MPLs show a larger cloud fraction at low altitudes. The difference in mean ice cloud top and base heights at the Manus and Nauru sites are all within 0.51 km, although differences are statistically significant. Mean ice cloud geometrical thickness agree to within 0.05 km at the Manus and Nauru sites. Larger differences exist at Darwin due to excessive degradation of the MPL output power during our sampling period. Both sets of observations show thicker clouds during the nighttime which may be real but could also be partially an artifact of the decreased signal-to-noise ratio during the daytime. The number of ice cloud layers per profile are also shown to be consistent after accounting for the difference in spatial resolution. For cloud optical depths, four different retrieval methods are compared, two for each set of observations. All products show that the majority of ice cloud optical depths ({approx}60%) fall below an optical depth of 0.2. For most comparisons all four retrievals agree to within the uncertainty intervals. We find that both CALIPSO retrievals agree best to ground-based optical depths when the lidar ratio in the latter is retrieved instead of set to a fixed value. Also thoroughly compared is the cloud properties for the subset of ice clouds which reside in the tropical tropopause layer (TTL).

  18. TH-A-19A-06: Site-Specific Comparison of Analytical and Monte Carlo Based Dose Calculations

    SciTech Connect

    Schuemann, J; Grassberger, C; Paganetti, H; Dowdell, S

    2014-06-15

    Purpose: To investigate the impact of complex patient geometries on the capability of analytical dose calculation algorithms to accurately predict dose distributions and to verify currently used uncertainty margins in proton therapy. Methods: Dose distributions predicted by an analytical pencilbeam algorithm were compared with Monte Carlo simulations (MCS) using TOPAS. 79 complete patient treatment plans were investigated for 7 disease sites (liver, prostate, breast, medulloblastoma spine and whole brain, lung and head and neck). A total of 508 individual passively scattered treatment fields were analyzed for field specific properties. Comparisons based on target coverage indices (EUD, D95, D90 and D50) were performed. Range differences were estimated for the distal position of the 90% dose level (R90) and the 50% dose level (R50). Two-dimensional distal dose surfaces were calculated and the root mean square differences (RMSD), average range difference (ARD) and average distal dose degradation (ADD), the distance between the distal position of the 80% and 20% dose levels (R80- R20), were analyzed. Results: We found target coverage indices calculated by TOPAS to generally be around 1–2% lower than predicted by the analytical algorithm. Differences in R90 predicted by TOPAS and the planning system can be larger than currently applied range margins in proton therapy for small regions distal to the target volume. We estimate new site-specific range margins (R90) for analytical dose calculations considering total range uncertainties and uncertainties from dose calculation alone based on the RMSD. Our results demonstrate that a reduction of currently used uncertainty margins is feasible for liver, prostate and whole brain fields even without introducing MC dose calculations. Conclusion: Analytical dose calculation algorithms predict dose distributions within clinical limits for more homogeneous patients sites (liver, prostate, whole brain). However, we recommend

  19. Experimental Comparison of two Active Vibration Control Approaches: Velocity Feedback and Negative Capacitance Shunt Damping

    NASA Technical Reports Server (NTRS)

    Beck, Benjamin; Schiller, Noah

    2013-01-01

    This paper outlines a direct, experimental comparison between two established active vibration control techniques. Active vibration control methods, many of which rely upon piezoelectric patches as actuators and/or sensors, have been widely studied, showing many advantages over passive techniques. However, few direct comparisons between different active vibration control methods have been made to determine the performance benefit of one method over another. For the comparison here, the first control method, velocity feedback, is implemented using four accelerometers that act as sensors along with an analog control circuit which drives a piezoelectric actuator. The second method, negative capacitance shunt damping, consists of a basic analog circuit which utilizes a single piezoelectric patch as both a sensor and actuator. Both of these control methods are implemented individually using the same piezoelectric actuator attached to a clamped Plexiglas window. To assess the performance of each control method, the spatially averaged velocity of the window is compared to an uncontrolled response.

  20. Comparison of (90)Y activity measurements in nuclear medicine in Germany.

    PubMed

    Kossert, Karsten; Bokeloh, Karen; Ehlers, Marion; Nähle, Ole; Scheibe, Olaf; Schwarz, Uwe; Thieme, Klaus

    2016-03-01

    In 2014, PTB and the company Eckert & Ziegler organized a national comparison exercise to determine the activity of a (90)Y solution. One aim of the comparison was to assess the measurement capability of hospitals and medical practices in Germany. P6-type vials were filled with aliquots of a radioactive (90)Y solution and then sent to 19 participants who were asked to measure the activity in the ampoules as well as in their own standard geometry using syringes. Most of the submitted results have a deviation of less than ±10% from the PTB reference activity when measured in the P6-type vials. The spread is somewhat larger when measured in a syringe geometry. The comparison revealed that some participants have difficulties in applying decay corrections and only a few participants were capable of estimating realistic measurement uncertainties. PMID:26597654

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    2014-01-01

    The ability to make behavioural inferences from skeletal remains is critical to understanding the lifestyles and activities of past human populations and extinct animals. Muscle attachment site (enthesis) morphology has long been assumed to reflect muscle strength and activity during life, but little experimental evidence exists to directly link activity patterns with muscle development and the morphology of their attachments to the skeleton. We used a mouse model to experimentally test how the level and type of activity influences forelimb muscle architecture of spinodeltoideus, acromiodeltoideus, and superficial pectoralis, bone growth rate and gross morphology of their insertion sites. Over an 11-week period, we collected data on activity levels in one control group and two experimental activity groups (running, climbing) of female wild-type mice. Our results show that both activity type and level increased bone growth rates influenced muscle architecture, including differences in potential muscular excursion (fibre length) and potential force production (physiological cross-sectional area). However, despite significant influences on muscle architecture and bone development, activity had no observable effect on enthesis morphology. These results suggest that the gross morphology of entheses is less reliable than internal bone structure for making inferences about an individual’s past behaviour. PMID:25467113

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

    PubMed

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

    2007-09-01

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

  4. Asymmetry of the active site loop conformation between subunits of glutamate-1-semialdehyde aminomutase in solution.

    PubMed

    Campanini, Barbara; Bettati, Stefano; di Salvo, Martino Luigi; Mozzarelli, Andrea; Contestabile, Roberto

    2013-01-01

    Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5'-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5'-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.

  5. A comparison of active adverse event surveillance systems worldwide.

    PubMed

    Huang, Yu-Lin; Moon, Jinhee; Segal, Jodi B

    2014-08-01

    Post-marketing drug surveillance for adverse drug events (ADEs) has typically relied on spontaneous reporting. Recently, regulatory agencies have turned their attention to more preemptive approaches that use existing data for surveillance. We conducted an environmental scan to identify active surveillance systems worldwide that use existing data for the detection of ADEs. We extracted data about the systems' structures, data, and functions. We synthesized the information across systems to identify common features of these systems. We identified nine active surveillance systems. Two systems are US based-the FDA Sentinel Initiative (including both the Mini-Sentinel Initiative and the Federal Partner Collaboration) and the Vaccine Safety Datalink (VSD); two are Canadian-the Canadian Network for Observational Drug Effect Studies (CNODES) and the Vaccine and Immunization Surveillance in Ontario (VISION); and two are European-the Exploring and Understanding Adverse Drug Reactions by Integrative Mining of Clinical Records and Biomedical Knowledge (EU-ADR) Alliance and the Vaccine Adverse Event Surveillance and Communication (VAESCO). Additionally, there is the Asian Pharmacoepidemiology Network (AsPEN) and the Shanghai Drug Monitoring and Evaluative System (SDMES). We identified two systems in the UK-the Vigilance and Risk Management of Medicines (VRMM) Division and the Drug Safety Research Unit (DSRU), an independent academic unit. These surveillance systems mostly use administrative claims or electronic medical records; most conduct pharmacovigilance on behalf of a regulatory agency. Either a common data model or a centralized model is used to access existing data. The systems have been built using national data alone or via partnership with other countries. However, active surveillance systems using existing data remain rare. North America and Europe have the most population coverage; with Asian countries making good advances. PMID:25022829

  6. Comparison of bactericidal and cytotoxic activities of trichogin analogs.

    PubMed

    Tavano, Regina; Malachin, Giulia; De Zotti, Marta; Peggion, Cristina; Biondi, Barbara; Formaggio, Fernando; Papini, Emanuele

    2016-03-01

    Peptaibiotics are a group of membrane active peptides of fungal origin. They typically contain α-aminoisobutyric acid (Aib; 1-letter code, U) and other non-coded residues (Toniolo and Brückner, 2009; Neumann et al., 2015; Benedett et al., 1982) [1], [2], [3] stabilizing their helical structure. Peptaibols are peptaibiotics carrying a 1, 2-aminoalcohol at the C-terminus. When a fatty acid chain (of 8-10 carbon atoms) is present at their N-terminus, they are called lipopeptaibols (Toniolo et al., 2001; Degenkolb et al., 2003) [4], [5]. We found (Tavano et al., 2015) [6] that the lipopeptaibol trichogin displays no antibacterial effects up to 64 µM, against both Gram(-) and Gram(+) bacteria, but kills tumor and healthy human cells via a mechanism requiring both the C-terminal primary alcohol group and the N-terminal n-octanoyl moiety, with EC50s around 4-5 µM. However, the substitution of single Gly residues with Lys strongly improves anti-Gram(+) activity (Tavano et al., 2015; De Zotti, Biondi, Park et al., 2012; De Zotti, Biondi, Peggion et al., 2012) [6], [7], [8]. To further characterize the activity of trichogin analogs as antibiotics and cytotoxic agents, we here manipulated the peptide helix amphipathicity by means of two different substitutions: (i) Aib to Leu (De Zotti et al., 2012) [7] or (ii) multiple Gly to Lys changes (Tavano et al., 2015; De Zotti, Biondi, Park et al., 2012; De Zotti, Biondi, Peggion, Formaggio et al., 2012; De Zotti, Biondi, Peggion, De Poli et al., 2012) [6], [7], [8], [9]. The antibacterial activity against four commensal or opportunistic bacterial species and the cytotoxicity against a panel of 9 healthy and tumor-derived eukaryotic cell types (including erythrocytes) are reported as MIC and EC50 (MTS - [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)]-2H-tetrazolium- reduction and LDH - lactate dehydrogenase - release assay). PMID:26862583

  7. Comparison of bactericidal and cytotoxic activities of trichogin analogs

    PubMed Central

    Tavano, Regina; Malachin, Giulia; De Zotti, Marta; Peggion, Cristina; Biondi, Barbara; Formaggio, Fernando; Papini, Emanuele

    2015-01-01

    Peptaibiotics are a group of membrane active peptides of fungal origin. They typically contain α-aminoisobutyric acid (Aib; 1-letter code, U) and other non-coded residues (Toniolo and Brückner, 2009; Neumann et al., 2015; Benedett et al., 1982) [1], [2], [3] stabilizing their helical structure. Peptaibols are peptaibiotics carrying a 1, 2-aminoalcohol at the C-terminus. When a fatty acid chain (of 8–10 carbon atoms) is present at their N-terminus, they are called lipopeptaibols (Toniolo et al., 2001; Degenkolb et al., 2003) [4], [5]. We found (Tavano et al., 2015) [6] that the lipopeptaibol trichogin displays no antibacterial effects up to 64 µM, against both Gram− and Gram+ bacteria, but kills tumor and healthy human cells via a mechanism requiring both the C-terminal primary alcohol group and the N-terminal n-octanoyl moiety, with EC50s around 4–5 µM. However, the substitution of single Gly residues with Lys strongly improves anti-Gram+ activity (Tavano et al., 2015; De Zotti, Biondi, Park et al., 2012; De Zotti, Biondi, Peggion et al., 2012) [6], [7], [8]. To further characterize the activity of trichogin analogs as antibiotics and cytotoxic agents, we here manipulated the peptide helix amphipathicity by means of two different substitutions: (i) Aib to Leu (De Zotti et al., 2012) [7] or (ii) multiple Gly to Lys changes (Tavano et al., 2015; De Zotti, Biondi, Park et al., 2012; De Zotti, Biondi, Peggion, Formaggio et al., 2012; De Zotti, Biondi, Peggion, De Poli et al., 2012) [6], [7], [8], [9]. The antibacterial activity against four commensal or opportunistic bacterial species and the cytotoxicity against a panel of 9 healthy and tumor-derived eukaryotic cell types (including erythrocytes) are reported as MIC and EC50 (MTS - [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)]-2H-tetrazolium- reduction and LDH - lactate dehydrogenase - release assay). PMID:26862583

  8. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity

    PubMed Central

    Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H.

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

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

    PubMed

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

    1975-10-10

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

  10. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity.

    PubMed

    Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

  11. Alternative poly(A) site utilization during adenovirus infection coincides with a decrease in the activity of a poly(A) site processing factor.

    PubMed Central

    Mann, K P; Weiss, E A; Nevins, J R

    1993-01-01

    The recognition and processing of a pre-mRNA to create a poly(A) addition site, a necessary step in mRNA biogenesis, can also be a regulatory event in instances in which the frequency of use of a poly(A) site varies. One such case is found during the course of an adenovirus infection. Five poly(A) sites are utilized within the major late transcription unit to produce more than 20 distinct mRNAs during the late phase of infection. The proximal half of the major late transcription unit is also expressed during the early phase of a viral infection. During this early phase of expression, the L1 poly(A) site is used three times more frequently than the L3 poly(A) site. In contrast, the L3 site is used three times more frequently than the L1 site during the late phase of infection. Recent experiments have suggested that the recognition of the poly(A) site GU-rich downstream element by the CF1 processing factor may be a rate-determining step in poly(A) site selection. We demonstrate that the interaction of CF1 with the L1 poly(A) site is less stable than the interaction of CF1 with the L3 poly(A) site. We also find that there is a substantial decrease in the level of CF1 activity when an adenovirus infection proceeds to the late phase. We suggest that this reduction in CF1 activity, coupled with the relative instability of the interaction with the L1 poly(A) site, contributes to the reduced use of the L1 poly(A) site during the late stage of an adenovirus infection. Images PMID:8384308

  12. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase*

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  13. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

    PubMed

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

  14. Comparison of the antioxidant activities of extra virgin olive oils.

    PubMed

    Lavelli, Vera

    2002-12-18

    The phenol content and antioxidant activity of extra virgin olive oils (EVOOs) differing in their origins and degradation degrees were studied. The o-diphenolic compounds typical of olive oil, namely, the oleuropein derivatives hydroxytyrosol (3',4'-dihydroxyphenylethanol, 3',4'-DHPEA), the dialdehydic form of elenolic acid linked to 3',4'-DHPEA (3',4'-DHPEA-EDA), and an isomer of oleuropein aglycon (3',4'-DHPEA-EA), were analyzed by HPLC. The antioxidant activity was studied by (a) the xanthine oxidase (XOD)/xanthine system, which generates superoxide radical and hydrogen peroxide; (b) the diaphorase (DIA)/NADH/juglone system, which generates superoxide radical and semiquinonic radical; and (c) the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) test. Results showed that EVOOs with a low degradation level (as evaluated by acidity, peroxide number, and spectroscopic indices K(232), K(270), and deltaK according to the EU Regulation) had a higher content of 3',4'-DHPEA-EDA and a lower content of 3',4'-DHPEA than oils having intermediate and advanced degradation levels. EVOOs with a low degradation degree were 3-5 times more efficient as DPPH scavengers and 2 times more efficient as inhibitors of the XOD-catalyzed reaction than oils with intermediate and advanced degradation levels. The DIA-catalyzed reaction was inhibited by EVOOs having low or intermediate degradation levels but not by the most degraded oils.

  15. Climatology of Warm Boundary Layer Clouds at the ARM SGP Site and Their Comparison to Models

    SciTech Connect

    Sengupta, Manajit; Clothiaux, Eugene E.; Ackerman, Thomas P.

    2004-12-01

    A four-year climatology (1997-2000) of warm boundary layer cloud properties is developed for the U.S. Department of Energy Atmospheric Radiation (ARM) Program Southern Great Plains (SGP) site. Parameters in the climatology include cloud liquid water path, cloud base height and surface solar flux. These parameters are retrieved from measurements produced by a dual-channel microwave radiometer, a millimeter-wave cloud radar, a micropulse lidar, a Belfort ceilometer, shortwave radiometers and atmospheric temperature profiles amalgamated from multiple sources, including radiosondes. No significant interannual differences are observed, but nighttime liquid water paths are consistently higher than daytime values. The summer months of June, July and August have the lowest liquid water paths and the highest cloud base heights. Model outputs of cloud liquid water paths from the European Center for Medium Range Weather Forecasting (ECMWF) model and the Early Eta Model for 104 Model Output Location Time Series (MOLTS) stations in the environs of the SGP central facility are compared to observations. The ECMWF and MOLTS mean and median liquid water paths are 3 and 4 times greater, respectively, than the observed values. The MOLTS data show lower liquid water paths in summer, which is consistent with observations, while the ECMWF data exhibit the opposite tendency. A parameterization of normalized cloud forcing that requires only cloud liquid water path and solar zenith angle is developed from the observations. The parameterization, which has a correlation coefficient of 0.81 with the observations, provides estimates of surface solar flux that are comparable to values obtained from explicit radiative transfer calculations based on plane-parallel theory. This parameterization is used to estimate the impact on the surface solar flux of differences in the liquid water paths between models and observations. Overall, there is a low bias of 50% in modeled normalized cloud forcing

  16. A comparison of multi-site daily rainfall downscaling techniques under Australian conditions

    NASA Astrophysics Data System (ADS)

    Frost, Andrew J.; Charles, Stephen P.; Timbal, Bertrand; Chiew, Francis H. S.; Mehrotra, R.; Nguyen, Kim C.; Chandler, Richard E.; McGregor, John L.; Fu, Guobin; Kirono, Dewi G. C.; Fernandez, Elodie; Kent, David M.

    2011-09-01

    SummarySix methods of downscaling GCM simulations to multi-site daily precipitation were applied to a set of 30 rain gauges located within South-Eastern Australia. The methods were tested at reproducing a range of statistics important within hydrological studies including inter-annual variability and spatial coherency using both NCEP/NCAR reanalysis and GCM predictors, thus testing the validity of GCM downscaled predictions. The methods evaluated, all having found application in Australia previously, are: (1) the dynamical downscaling Conformal-Cubic Atmospheric Model (CCAM) of McGregor (2005); the historical data based (2) Scaling method applied by Chiew et al. (2009) and (3) Analogue method of Timbal (2004); and three stochastic methods, (4) the GLIMCLIM (Generalised Linear Model for daily Climate time series) software package ( Chandler, 2002), (5) the Non-homogeneous Hidden Markov Model (NHMM) of Charles et al. (1999), and (6) the modified Markov model-kernel probability density estimation (MMM-KDE) downscaling technique of Mehrotra and Sharma (2007). The results showed that the simple Scaling approach provided relatively robust results for a range of statistics when GCM forcing data was used, and was therefore recommended for regional water availability and planning studies (subject to certain limitations as mentioned in conclusion section). The stochastic methods better capture changes to a fuller range of rainfall statistics and are recommended for detailed catchment modelling studies. In particular, the stochastic methods show better results for daily extreme rainfall (e.g. flooding/low flow) as the simulations are not based purely on temporal/spatial rainfall patterns observed in the past, and a hybrid GLIMCLIM occurrence-KDE amounts model is recommended based on performance for individual statistics. For GCM downscaled simulations, biases in annual mean and standard deviation of ±5% and -30% were observed typically, and no single model performed well

  17. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    SciTech Connect

    Parker, Shane M.; Shiozaki, Toru

    2014-12-07

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μE{sub h} or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation.

  18. A Frontier Molecular Orbital determination of the active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.

    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 a dispersed metal catalysts. The basis for these calculations is the reported finding that a large number of catalyzed reactions take place on single atom active sites on the metal surface. Thus, these sites can be considered as surface complexes made up of the central active atom surrounded by near-neighbor metal atom ``ligands`` with localized surface orbitals perturbed only by these ``ligands``. These ``complexes`` are based on a twelve coordinate species with the ``ligands`` attached to the t{sub 2g} orbitals and the coordinate axes coincident with the direction of the e{sub g} orbitals on the central atom. 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.

  19. A Frontier Molecular Orbital determination of the active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.

    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 a dispersed metal catalysts. The basis for these calculations is the reported finding that a large number of catalyzed reactions take place on single atom active sites on the metal surface. Thus, these sites can be considered as surface complexes made up of the central active atom surrounded by near-neighbor metal atom ligands'' with localized surface orbitals perturbed only by these ligands''. These complexes'' are based on a twelve coordinate species with the ligands'' attached to the t{sub 2g} orbitals and the coordinate axes coincident with the direction of the e{sub g} orbitals on the central atom. 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.

  20. Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome

    PubMed Central

    Lebailly, Elise; Pages, Carine; Cornet, Francois; Cosentino Lagomarsino, Marco

    2016-01-01

    Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle. PMID:27171414