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Sample records for active site compared

  1. Ionospheric plasma deterioration in the area of enhanced seismic activity as compared to antipodal sites far from seismicity

    NASA Astrophysics Data System (ADS)

    Gulyaeva, Tamara; Arikan, Feza; Poustovalova, Ljubov; Stanislawska, Iwona

    2016-07-01

    The early magnetogram records from two nearly antipodal sites at Greenwich and Melbourne corresponding to the activity level at the invariant magnetic latitude of 50 deg give a long series of geomagnetic aa indices since 1868. The aa index derived from magnetic perturbation values at only two observatories (as distinct from the planetary ap index) experiences larger extreme values if either input site is well situated to the overhead ionospheric and/or field aligned current systems producing the magnetic storm effects. Analysis of the earthquakes catalogues since 1914 has shown the area of the peak global earthquake occurrence in the Pacific Ocean southwards from the magnetic equator, and, in particular, at Australia. In the present study the ionospheric critical frequency, foF2, is analyzed from the ionosonde measurements at the nearby observatories, Canberra and Slough (Chilton), and Moscow (control site) since 1944 to 2015. The daily-hourly-annual percentage occurrence of positive ionospheric W index (pW+) and negative index (pW-) is determined. It is found that the ionospheric plasma depletion pW- of the instant foF2 as compared to the monthly median is well correlated to the aa index at all three sites but the positive storm signatures show drastic difference at Canberra (no correlation of pW+ with aa index) as compared to two other sites where the high correlation is found of the ionospheric plasma density enhancement with the geomagnetic activity. A possible suppression of the enhanced ionospheric variability over the region of intense seismicity is discussed in the paper. This study is supported by TUBITAK EEEAG 115E915.

  2. 1H-NMR comparative study of the active site in shark (Galeorhinus japonicus), horse, and sperm whale deoxy myoglobins.

    PubMed

    Yamamoto, Y; Iwafune, K; Chûjô, R; Inoue, Y; Imai, K; Suzuki, T

    1992-09-01

    1H-NMR spectra of deoxy myoglobins (Mbs) from shark (Galeorhinus japonicus), horse, and sperm whale have been studied to gain insights into their active site structure. It has been demonstrated for the first time that nuclear Overhauser effect (NOE) can be observed between heme peripheral side-chain proton resonances of these paramagnetic complexes. Val-E11 methyl and His-F8 C delta H proton resonances of these Mbs were also assigned from the characteristic shift and line width. The hyperfine shift of the former resonance was used to calculate the magnetic anisotropy of the protein. The shift analysis of the latter resonance, together with the previously assigned His-F8 N delta H proton resonance, revealed that the strain on the Fe-N epsilon bond is in the order horse Mb approximately whale Mb < shark Mb and that the hydrogen bond strength of the His-F8 N delta H proton to the main-chain carbonyl oxygen in the preceding turn of the F helix is in the order shark Mb < horse Mb < whale Mb. Weaker Feporphyrin interaction in shark Mb was manifested in a smaller shift of the heme methyl proton resonance and appears to result from distortion of the coordination geometry in this Mb. Larger strain on the Fe-N epsilon bond in shark Mb should be to some extent attributed to its lowered O2 affinity (P50 = 1.1 mmHg at 20 degrees C), compared to whale and horse Mbs.

  3. Angiotensin-converting enzyme-2 (ACE2): comparative modeling of the active site, specificity requirements, and chloride dependence.

    PubMed

    Guy, Jodie L; Jackson, Richard M; Acharya, K Ravi; Sturrock, Edward D; Hooper, Nigel M; Turner, Anthony J

    2003-11-18

    Angiotensin-converting enzyme 2 (ACE2), a homologue of ACE, represents a new and potentially important target in cardio-renal disease. A model of the active site of ACE2, based on the crystal structure of testicular ACE, has been developed and indicates that the catalytic mechanism of ACE2 resembles that of ACE. Structural differences exist between the active site of ACE (dipeptidyl carboxypeptidase) and ACE2 (carboxypeptidase) that are responsible for the differences in specificity. The main differences occur in the ligand-binding pockets, particularly at the S2' subsite and in the binding of the peptide carboxy-terminus. The model explains why the classical ACE inhibitor lisinopril is unable to bind to ACE2. On the basis of the ability of ACE2 to cleave a variety of biologically active peptides, a consensus sequence of Pro-X-Pro-hydrophobic/basic for the protease specificity of ACE2 has been defined that is supported by the ACE2 model. The dipeptide, Pro-Phe, completely inhibits ACE2 activity at 180 microM with angiotensin II as the substrate. As with ACE, the chloride dependence of ACE2 is substrate-specific such that the hydrolysis of angiotensin I and the synthetic peptide substrate, Mca-APK(Dnp), are activated in the presence of chloride ions, whereas the cleavage of angiotensin II is inhibited. The ACE2 model is also suggestive of a possible mechanism for chloride activation. The structural insights provided by these analyses for the differences in inhibition pattern and substrate specificity among ACE and its homologue ACE2 and for the chloride dependence of ACE/ACE2 activity are valuable in understanding the function and regulation of ACE2.

  4. Influence of Human Activity Patterns, particle composition, and residential air exchange rates on modeled distributions of PM 2.5 exposure compared with central-site monitoring data

    EPA Science Inventory

    Central-site monitors do not account for factors such as outdoor-to-indoor transport and human activity patterns that influence personal exposures to ambient fine-particulate matter (PM2.5). We describe and compare different ambient PM2.5 exposure estimation...

  5. List 9 - Active CERCLIS Sites:

    EPA Pesticide Factsheets

    The List 9 displays the sequence of activities undertaken at active CERCLIS sites. An active site is one at which site assessment, removal, remedial, enforcement, cost recovery, or oversight activities are being planned or conducted.

  6. Comparative analysis of amino acid composition in the active site of nirk gene encoding copper-containing nitrite reductase (CuNiR) in bacterial spp.

    PubMed

    Adhikari, Utpal Kumar; Rahman, M Mizanur

    2017-04-01

    The nirk gene encoding the copper-containing nitrite reductase (CuNiR), a key catalytic enzyme in the environmental denitrification process that helps to produce nitric oxide from nitrite. The molecular mechanism of denitrification process is definitely complex and in this case a theoretical investigation has been conducted to know the sequence information and amino acid composition of the active site of CuNiR enzyme using various Bioinformatics tools. 10 Fasta formatted sequences were retrieved from the NCBI database and the domain and disordered regions identification and phylogenetic analyses were done on these sequences. The comparative modeling of protein was performed through Modeller 9v14 program and visualized by PyMOL tools. Validated protein models were deposited in the Protein Model Database (PMDB) (PMDB id: PM0080150 to PM0080159). Active sites of nirk encoding CuNiR enzyme were identified by Castp server. The PROCHECK showed significant scores for four protein models in the most favored regions of the Ramachandran plot. Active sites and cavities prediction exhibited that the amino acid, namely Glycine, Alanine, Histidine, Aspartic acid, Glutamic acid, Threonine, and Glutamine were common in four predicted protein models. The present in silico study anticipates that active site analyses result will pave the way for further research on the complex denitrification mechanism of the selected species in the experimental laboratory.

  7. Comparative residue interaction analysis (CoRIA): a 3D-QSAR approach to explore the binding contributions of active site residues with ligands

    NASA Astrophysics Data System (ADS)

    Datar, Prasanna A.; Khedkar, Santosh A.; Malde, Alpeshkumar K.; Coutinho, Evans C.

    2006-06-01

    A novel approach termed comparative residue-interaction analysis (CoRIA), emphasizing the trends and principles of QSAR in a ligand-receptor environment has been developed to analyze and predict the binding affinity of enzyme inhibitors. To test this new approach, a training set of 36 COX-2 inhibitors belonging to nine families was selected. The putative binding (bioactive) conformations of inhibitors in the COX-2 active site were searched using the program DOCK. The docked configurations were further refined by a combination of Monte Carlo and simulated annealing methods with the Affinity program. The non-bonded interaction energies of the inhibitors with the individual amino acid residues in the active site were then computed. These interaction energies, plus specific terms describing the thermodynamics of ligand-enzyme binding, were correlated to the biological activity with G/PLS. The various QSAR models obtained were validated internally by cross validation and boot strapping, and externally using a test set of 13 molecules. The QSAR models developed on the CoRIA formalism were robust with good r 2, q 2 and r pred 2 values. The major highlights of the method are: adaptation of the QSAR formalism in a receptor setting to answer both the type (qualitative) and the extent (quantitative) of ligand-receptor binding, and use of descriptors that account for the complete thermodynamics of the ligand-receptor binding. The CoRIA approach can be used to identify crucial interactions of inhibitors with the enzyme at the residue level, which can be gainfully exploited in optimizing the inhibitory activity of ligands. Furthermore, it can be used with advantage to guide point mutation studies. As regards the COX-2 dataset, the CoRIA approach shows that improving Coulombic interaction with Pro528 and reducing van der Waals interaction with Tyr385 will improve the binding affinity of inhibitors.

  8. Validity of wrist worn accelerometers and comparability between hip and wrist placement sites in estimating physical activity behaviour in preschool children.

    PubMed

    Hislop, Jane; Palmer, Nicole; Anand, Priya; Aldin, Tara

    2016-09-21

    Wrist-worn accelerometers can increase compliance with wearing accelerometers, however, several large scale studies continue to use hip-worn accelerometers and it is unclear how comparable data is from the two sites. The study aims were: to investigate agreement between wrist- and hip- worn accelerometers and to determine the validity of Johansson et al cut-points for wrist worn accelerometers in preschool children. A sample of 32 preschool children (21 boys, 4.2 (0.5) years, BMI 16.6 (1.1)) were videoed wearing GT3X+  accelerometers on their wrist and hip while they engaged in 1 h of free-play in their nursery. Children's activity were coded using, the children's activity rating scale (CARS): with CARS, level 1 'sedentary' and levels 2-5 were classified as time spent in total physical activity (TPA). Accelerometry data were processed using Johansson et al cut-points for the wrist data and Evenson et al cut-points for the hip data, into time spent in different intensities of physical activity (PA). The mean counts per minute (cpm) from the hip and wrist were compared. There was a strong correlation between the hip and wrist cpm (r  =  0.81, p  <  0.01) and total count data (r  =  0.83 p  <  0.01), however there was a large systematic bias with wide limits of agreement. Good agreement (mean difference (LOA) 1.1 (-9.9, 12.1) was found between the CARS estimate of TPA (29.5 (10.4) mins) and the wrist estimate, using the Johansson et al cut points (28.4 (9.8) mins). There was also a reasonable agreement between the hip estimates with the Evenson et al cut-points and Johansson et al estimate (mean difference (LOA):6.3 (-8.8, 21.4) mins. In conclusion, the findings suggest that the Johansson et al (2013 Pediatr. Obes. 10 1-6) cut-points applied to wrist worn accelerometers provides a valid estimate of TPA in preschool children and have reasonable agreement with Evenson et al cut-points applied to hip accelerometers.

  9. 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.

  10. Normal Modes Expose Active Sites in Enzymes

    PubMed Central

    Glantz-Gashai, Yitav; Samson, Abraham O.

    2016-01-01

    Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

  11. Comparability of skin temperatures from three sites on the hand.

    PubMed

    Duckro, P N; Schultz, K; Shaffer, F

    1986-12-01

    Skin temperature from three recording sites (web dorsum and two digital sites) on one hand were compared over a 30-minute period during which room temperature was raised from 66 degrees to 80 degrees F causing skin temperatures to range from 78.5 degrees to 92.3 degrees F. The web dorsum remained significantly warmer than either digital site at the lowest skin temperatures; no significant inter-site differences were seen as skin temperature gradually increased. These findings imply that none of the three sites is more advantageous than the others when recording hand temperature values around or above 85 degrees F. However, at the lower levels of skin temperature, there is a "floor" below which the web dorsum is no longer a sensitive indicator. More generally, basic data of this type provide a necessary but often absent foundation for routine practice of clinical biofeedback.

  12. Validated ligand mapping of ACE active site

    NASA Astrophysics Data System (ADS)

    Kuster, Daniel J.; Marshall, Garland R.

    2005-08-01

    Crystal structures of angiotensin-converting enzyme (ACE) complexed with three inhibitors (lisinopril, captopril, enalapril) provided experimental data for testing the validity of a prior active site model predicting the bound conformation of the inhibitors. The ACE active site model - predicted over 18 years ago using a series of potent ACE inhibitors of diverse chemical structure - was recreated using published data and commercial software. Comparison between the predicted structures of the three inhibitors bound to the active site of ACE and those determined experimentally yielded root mean square deviation (RMSD) values of 0.43-0.81 Å, among the distances defining the active site map. The bound conformations of the chemically relevant atoms were accurately deduced from the geometry of ligands, applying the assumption that the geometry of the active site groups responsible for binding and catalysis of amide hydrolysis was constrained. The mapping of bound inhibitors at the ACE active site was validated for known experimental compounds, so that the constrained conformational search methodology may be applied with confidence when no experimentally determined structure of the enzyme yet exists, but potent, diverse inhibitors are available.

  13. The MicrobesOnline Web site for comparative genomics

    SciTech Connect

    Alm, Eric J.; Huang, Katherine H.; Price, Morgan N.; Koche,Richard P.; Keller, Keith; Dubchak, Inna L.; Arkin, Adam P.

    2004-11-05

    At present, hundreds of microbial genomes have been sequenced, and hundreds more are currently in the pipeline. The Virtual Institute for Microbial Stress and Survival has developed a publicly available suite of Web-based comparative genomic tools (http://www.microbesonline.org) designed to facilitate multispecies comparison among prokaryotes. Highlights of the Microbes Online Web site include operon and regulon predictions, a multispecies genome browser, a multispecies Gene Ontology browser, a comparative KEGG metabolic pathway viewer, a Bioinformatics Workbench for in-depth sequence analysis, and Gene Carts that allow users to save genes of interest for further study while they browse. In addition, we provide an interface for genome annotation, which like all of the tools reported here, is freely available to the scientific community.

  14. The aggregate site frequency spectrum for comparative population genomic inference.

    PubMed

    Xue, Alexander T; Hickerson, Michael J

    2015-12-01

    Understanding how assemblages of species responded to past climate change is a central goal of comparative phylogeography and comparative population genomics, an endeavour that has increasing potential to integrate with community ecology. New sequencing technology now provides the potential to perform complex demographic inference at unprecedented resolution across assemblages of nonmodel species. To this end, we introduce the aggregate site frequency spectrum (aSFS), an expansion of the site frequency spectrum to use single nucleotide polymorphism (SNP) data sets collected from multiple, co-distributed species for assemblage-level demographic inference. We describe how the aSFS is constructed over an arbitrary number of independent population samples and then demonstrate how the aSFS can differentiate various multispecies demographic histories under a wide range of sampling configurations while allowing effective population sizes and expansion magnitudes to vary independently. We subsequently couple the aSFS with a hierarchical approximate Bayesian computation (hABC) framework to estimate degree of temporal synchronicity in expansion times across taxa, including an empirical demonstration with a data set consisting of five populations of the threespine stickleback (Gasterosteus aculeatus). Corroborating what is generally understood about the recent postglacial origins of these populations, the joint aSFS/hABC analysis strongly suggests that the stickleback data are most consistent with synchronous expansion after the Last Glacial Maximum (posterior probability = 0.99). The aSFS will have general application for multilevel statistical frameworks to test models involving assemblages and/or communities, and as large-scale SNP data from nonmodel species become routine, the aSFS expands the potential for powerful next-generation comparative population genomic inference.

  15. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

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

  16. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

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

  17. Comparative toxicology of laboratory organisms for assessing hazardous waste sites

    SciTech Connect

    Miller, W.E.; Peterson, S.A.; Greene, J.C.; Callahan, C.A.

    1985-01-01

    Multi-media/multi-trophic level bioassays have been proposed to determine the extent and severity of environmental contamination at hazardous waste sites. Comparative toxicological profiles for algae (Selenastrum capricornutum), daphnia (Daphnia magna), earthworms (Eisenia foetida), microbes (Photobacterium fisherii, mixed sewage microorganisms) and plants; wheat Stephens, (Triticum aestivum), lettuce, butter crunch, (Lactuca sativa L.) radish, Cherry Belle, (Raphanus sativa L.), red clover, Kenland, (Trifolium pratense L.) and cucumber, Spartan Valor, (Cucumis sativa L.) are presented for selected heavy metals, herbicides and insecticides. Specific chemical EC/sub 50/ values are presented for each test organism. Differences in standard deviations were compared between each individual test organism, as well as for the chemical subgroup assayed. Algae and daphnia are the most sensitive test organisms to heavy metals and insecticides followed in order of decreasing sensitivity by Microtox (Photobacterium fisherii), DO depletion rate, seed germination and earthworms. Higher plants were most sensitive to 2,4-D, (2,4-Dichlorophenoxy acetic acid) followed by algae, Microtox, daphnia and earthworms. Differences in toxicity of 2,4-D chemical formulations and commercial sources of insecticides were observed with algae and daphia tests.

  18. Comparative study of biochemical and immunological biomarkers in three marine bivalves exposed at a polluted site.

    PubMed

    Cotou, Efthimia; Tsangaris, Catherine; Henry, Morgane

    2013-03-01

    A battery of biochemical and immunological biomarkers used for pollution assessment were measured for first time in the clams Venus verrucosa and Callista chione and were compared with those of the mussel Mytilus galloprovincialis, a well-established indicator organism utilized in numerous environmental monitoring programs. Clams and mussel were transplanted at a polluted and a reference site or maintained at the laboratory. Among biochemical biomarkers, acetylcholinesterase did not differ at the polluted site in all species, but there was a significant difference between the mussel and the clams, glutathione S-transferase showed a clear inhibition at the polluted site in all species and a significant difference between the two clams was also indicated, while catalase activities were increased only in V. verrucosa at the polluted site and not in mussel or the other clam. Immunological biomarkers responses were also pronounced at the polluted site. Lysozyme activity was species-dependent whereas respiratory burst activity measured as luminol-dependent chemiluminescence (CL) was site and stimulus dependent, and it was evident in M. galloprovincialis and V. verrucosa and not in C. chione. Further investigation focused on biochemical and immunological biomarkers related with the oxidative mechanisms in clams will strengthen and expand their use as bioindicators for pollution assessment.

  19. 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.

  20. [Structural regularities in activated cleavage sites of thrombin receptors].

    PubMed

    Mikhaĭlik, I V; Verevka, S V

    1999-01-01

    Comparison of thrombin receptors activation splitting sites sequences testifies to their similarity both in activation splitting sites of protein precursors and protein proteinase inhibitors reactive sites. In all these sites corresponded to effectory sites P2'-positions are placed by hydrophobic amino-acids only. The regularity defined conforms with previous thesis about the role of effectory S2'-site in regulation of the processes mediated by serine proteinases.

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

    PubMed

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

    2007-06-26

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

  3. Comparative hydrogen-deuterium exchange for a mesophilic vs thermophilic dihydrofolate reductase at 25 °C: identification of a single active site region with enhanced flexibility in the mesophilic protein.

    PubMed

    Oyeyemi, Olayinka A; Sours, Kevin M; Lee, Thomas; Kohen, Amnon; Resing, Katheryn A; Ahn, Natalie G; Klinman, Judith P

    2011-09-27

    The technique of hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been applied to a mesophilic (E. coli) dihydrofolate reductase under conditions that allow direct comparison to a thermophilic (B. stearothermophilus) ortholog, Ec-DHFR and Bs-DHFR, respectively. The analysis of hydrogen-deuterium exchange patterns within proteolytically derived peptides allows spatial resolution, while requiring a series of controls to compare orthologous proteins with only ca. 40% sequence identity. These controls include the determination of primary structure effects on intrinsic rate constants for HDX as well as the use of existing 3-dimensional structures to evaluate the distance of each backbone amide hydrogen to the protein surface. Only a single peptide from the Ec-DHFR is found to be substantially more flexible than the Bs-DHFR at 25 °C in a region located within the protein interior at the intersection of the cofactor and substrate-binding sites. The surrounding regions of the enzyme are either unchanged or more flexible in the thermophilic DHFR from B. stearothermophilus. The region with increased flexibility in Ec-DHFR corresponds to one of two regions previously proposed to control the enthalpic barrier for hydride transfer in Bs-DHFR [Oyeyemi et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 10074].

  4. Face the Edges: Catalytic Active Sites of Nanomaterials

    PubMed Central

    Ni, Bing

    2015-01-01

    Edges are special sites in nanomaterials. The atoms residing on the edges have different environments compared to those in other parts of a nanomaterial and, therefore, they may have different properties. Here, recent progress in nanomaterial fields is summarized from the viewpoint of the edges. Typically, edge sites in MoS2 or metals, other than surface atoms, can perform as active centers for catalytic reactions, so the method to enhance performance lies in the optimization of the edge structures. The edges of multicomponent interfaces present even more possibilities to enhance the activities of nanomaterials. Nanoframes and ultrathin nanowires have similarities to conventional edges of nanoparticles, the application of which as catalysts can help to reduce the use of costly materials. Looking beyond this, the edge structures of graphene are also essential for their properties. In short, the edge structure can influence many properties of materials. PMID:27980960

  5. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  6. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

  7. Activities in dementia care: A comparative assessment of activity types.

    PubMed

    Lokon, Elizabeth; Sauer, Philip E; Li, Yue

    2016-12-05

    This exploratory study compares the impact of five activity types on the well-being of institutionalized people with dementia: the intergenerational art program Opening Minds through Art, art and music therapies, creative activities, non-creative activities, and no activities at all. We validated the Scripps Modified Greater Cincinnati Chapter Well-Being Observational Tool, and used that instrument to systematically observe N = 67 people with dementia as they participated in different activity types. People with dementia showed the highest well-being scores during Opening Minds through Art compared to all other activities. No significant well-being differences were found between creative activities led by licensed art/music therapist versus regular activity staff. Furthermore, no significant well-being differences were found between creative and non-creative activities that were both led by regular activity staff. Overall, people with dementia benefit from participating in activities, regardless of the type (creative or non-creative), or who conducts them (licensed therapists or activity staff). However, in order for people with dementia to reach significantly high levels of overall well-being, we recommend that activities are specifically designed for people with dementia and incorporate a 1:1 ratio between people with dementia and well-trained volunteers/staff members.

  8. Active sites of thioredoxin reductases: why selenoproteins?

    PubMed

    Gromer, Stephan; Johansson, Linda; Bauer, Holger; Arscott, L David; Rauch, Susanne; Ballou, David P; Williams, Charles H; Schirmer, R Heiner; Arnér, Elias S J

    2003-10-28

    Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

  9. 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.

  10. Mutations inducing an active-site aperture in Rhizobium sp. sucrose isomerase confer hydrolytic activity.

    PubMed

    Lipski, Alexandra; Watzlawick, Hildegard; Ravaud, Stéphanie; Robert, Xavier; Rhimi, Moez; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2013-02-01

    Sucrose isomerase is an enzyme that catalyzes the production of sucrose isomers of high biotechnological and pharmaceutical interest. Owing to the complexity of the chemical synthesis of these isomers, isomaltulose and trehalulose, enzymatic conversion remains the preferred method for obtaining these products. Depending on the microbial source, the ratio of the sucrose-isomer products varies significantly. In studies aimed at understanding and explaining the underlying molecular mechanisms of these reactions, mutations obtained using a random-mutagenesis approach displayed a major hydrolytic activity. Two of these variants, R284C and F164L, of sucrose isomerase from Rhizobium sp. were therefore crystallized and their crystal structures were determined. The three-dimensional structures of these mutants allowed the identification of the molecular determinants that favour hydrolytic activity compared with transferase activity. Substantial conformational changes resulting in an active-site opening were observed, as were changes in the pattern of water molecules bordering the active-site region.

  11. Comparative Effectiveness Research Using Observational Data: Active Comparators to Emulate Target Trials with Inactive Comparators

    PubMed Central

    Huitfeldt, Anders; Hernan, Miguel A.; Kalager, Mette; Robins, James M.

    2016-01-01

    Introduction: Because a comparison of noninitiators and initiators of treatment may be hopelessly confounded, guidelines for the conduct of observational research often recommend using an “active” comparator group consisting of people who initiate a treatment other than the medication of interest. In this paper, we discuss the conditions under which this approach is valid if the goal is to emulate a trial with an inactive comparator. Identification of Effects: We provide conditions under which a target trial in a subpopulation can be validly emulated from observational data, using an active comparator that is known or believed to be inactive for the outcome of interest. The average treatment effect in the population as a whole is not identified, but under certain conditions this approach can be used to emulate a trial in the subset of individuals who were treated with the treatment of interest, in the subset of individuals who were treated with the treatment of interest but not with the comparator, or in the subset of individuals who were treated with both the treatment of interest and the active comparator. The Plausibility of the Comparability Conditions: We discuss whether the required conditions can be expected to hold in pharmacoepidemiologic research, with a particular focus on whether the conditions are plausible in situations where the standard analysis fails due to unmeasured confounding by access to health care or health seeking behaviors. Discussion: The conditions discussed in this paper may at best be approximately true. Investigators using active comparator designs to emulate trials with inactive comparators should exercise caution. PMID:27891526

  12. Comparative Sensitivity Analysis of Muscle Activation Dynamics

    PubMed Central

    Rockenfeller, Robert; Günther, Michael; Schmitt, Syn; Götz, Thomas

    2015-01-01

    We mathematically compared two models of mammalian striated muscle activation dynamics proposed by Hatze and Zajac. Both models are representative for a broad variety of biomechanical models formulated as ordinary differential equations (ODEs). These models incorporate parameters that directly represent known physiological properties. Other parameters have been introduced to reproduce empirical observations. We used sensitivity analysis to investigate the influence of model parameters on the ODE solutions. In addition, we expanded an existing approach to treating initial conditions as parameters and to calculating second-order sensitivities. Furthermore, we used a global sensitivity analysis approach to include finite ranges of parameter values. Hence, a theoretician striving for model reduction could use the method for identifying particularly low sensitivities to detect superfluous parameters. An experimenter could use it for identifying particularly high sensitivities to improve parameter estimation. Hatze's nonlinear model incorporates some parameters to which activation dynamics is clearly more sensitive than to any parameter in Zajac's linear model. Other than Zajac's model, Hatze's model can, however, reproduce measured shifts in optimal muscle length with varied muscle activity. Accordingly we extracted a specific parameter set for Hatze's model that combines best with a particular muscle force-length relation. PMID:26417379

  13. 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.

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

    PubMed

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

    2016-08-01

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

  15. 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.

  16. 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.

  17. 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.

  18. Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex

    PubMed Central

    Lim, Sung In; Yang, Byungseop; Jung, Younghan; Cha, Jaehyun; Cho, Jinhwan; Choi, Eun-Sil; Kim, Yong Hwan; Kwon, Inchan

    2016-01-01

    Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other. PMID:28004799

  19. Perspective: On the active site model in computational catalyst screening

    NASA Astrophysics Data System (ADS)

    Reuter, Karsten; Plaisance, Craig P.; Oberhofer, Harald; Andersen, Mie

    2017-01-01

    First-principles screening approaches exploiting energy trends in surface adsorption represent an unparalleled success story in recent computational catalysis research. Here we argue that our still limited understanding of the structure of active sites is one of the major bottlenecks towards an ever extended and reliable use of such computational screening for catalyst discovery. For low-index transition metal surfaces, the prevalently chosen high-symmetry (terrace and step) sites offered by the nominal bulk-truncated crystal lattice might be justified. For more complex surfaces and composite catalyst materials, computational screening studies will need to actively embrace a considerable uncertainty with respect to what truly are the active sites. By systematically exploring the space of possible active site motifs, such studies might eventually contribute towards a targeted design of optimized sites in future catalysts.

  20. Diffusional correlations among multiple active sites in a single enzyme.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

    Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

  1. Robotics at Savannah River site: activity report

    SciTech Connect

    Byrd, J.S.

    1984-09-01

    The objectives of the Robotics Technology Group at the Savannah River Laboratory are to employ modern industrial robots and to develop unique automation and robotic systems to enhance process operations at the Savannah River site (SRP and SRL). The incentives are to improve safety, reduce personnel radiation exposure, improve product quality and productivity, and to reduce operating costs. During the past year robotic systems have been installed to fill chemical dilution vials in a SRP laboratory at 772-F and remove radioactive waste materials in the SRL Californium Production Facility at 773-A. A robotic system to lubricate an extrusion press has been developed and demonstrated in the SRL robotics laboratory and is scheduled for installation at the 321-M fuel fabrication area. A mobile robot was employed by SRP for a radiation monitoring task at a waste tank top in H-Area. Several other robots are installed in the SRL robotics laboratories and application development programs are underway. The status of these applications is presented in this report.

  2. Comparative approaches to siting low-level radioactive waste disposal facilities

    SciTech Connect

    Newberry, W.F.

    1994-07-01

    This report describes activities in nine States to select site locations for new disposal facilities for low-level radioactive waste. These nine States have completed processes leading to identification of specific site locations for onsite investigations. For each State, the status, legal and regulatory framework, site criteria, and site selection process are described. In most cases, States and compact regions decided to assign responsibility for site selection to agencies of government and to use top-down mapping methods for site selection. The report discusses quantitative and qualitative techniques used in applying top-down screenings, various approaches for delineating units of land for comparison, issues involved in excluding land from further consideration, and different positions taken by the siting organizations in considering public acceptance, land use, and land availability as factors in site selection.

  3. 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.

  4. Community Update on Site Activities, July 19, 2013

    EPA Pesticide Factsheets

    In an effort to engage and inform community members interested in the New Bedford Harbor Superfund Site cleanup, EPA will be issuing periodic topic-based fact sheets that will provide background information and updates about ongoing activities.

  5. A comparative study between the fluxes of trace elements in bulk atmospheric deposition at industrial, urban, traffic, and rural sites.

    PubMed

    Fernández-Olmo, I; Puente, M; Irabien, A

    2015-09-01

    The input of trace elements via atmospheric deposition towards industrial, urban, traffic, and rural areas is quite different and depends on the intensity of the anthropogenic activity. A comparative study between the element deposition fluxes in four sampling sites (industrial, urban, traffic, and rural) of the Cantabria region (northern Spain) has been performed. Sampling was carried out monthly using a bulk (funnel bottle) sampler. The trace elements, As, Cd, Cr, Cu, Mn, Mo, Ni, Pb, Ti, Zn, and V, were determined in the water soluble and insoluble fractions of bulk deposition samples. The element deposition fluxes at the rural, urban, and traffic sites followed a similar order (Zn > Mn> > Cu ≈ Ti > Pb > V ≈ Cr > Ni> > As ≈ Mo > Cd). The most enriched elements were Cd, Zn, and Cu, while V, Ni, and Cr were less enriched. An extremely high deposition of Mn was found at the industrial site, leading to high enrichment factor values, resulting from the presence of a ferro-manganese/silico-manganese production plant in the vicinity of the sampling site. Important differences were found in the element solubilities in the studied sites; the element solubilities were higher at the traffic and rural sites, and lower at the urban and industrial sites. For all sites, Zn and Cd were the most soluble elements, whereas Cr and Ti were less soluble. The inter-site correlation coefficients for each element were calculated to assess the differences between the sites. The rural and traffic sites showed some similarities in the sources of trace elements; however, the sources of these elements at the industrial and rural sites were quite different. Additionally, the element fluxes measured in the insoluble fraction of the bulk atmospheric deposition exhibited a good correlation with the daily traffic volume at the traffic site.

  6. 10 CFR 960.3-2-2-3 - Comparative evaluation of all sites proposed for nomination.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 4 2012-01-01 2012-01-01 false Comparative evaluation of all sites proposed for nomination. 960.3-2-2-3 Section 960.3-2-2-3 Energy DEPARTMENT OF ENERGY GENERAL GUIDELINES FOR THE PRELIMINARY SCREENING OF POTENTIAL SITES FOR A NUCLEAR WASTE REPOSITORY Implementation Guidelines §...

  7. 10 CFR 960.3-2-2-3 - Comparative evaluation of all sites proposed for nomination.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 4 2011-01-01 2011-01-01 false Comparative evaluation of all sites proposed for nomination. 960.3-2-2-3 Section 960.3-2-2-3 Energy DEPARTMENT OF ENERGY GENERAL GUIDELINES FOR THE PRELIMINARY SCREENING OF POTENTIAL SITES FOR A NUCLEAR WASTE REPOSITORY Implementation Guidelines §...

  8. 10 CFR 960.3-2-2-3 - Comparative evaluation of all sites proposed for nomination.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 4 2013-01-01 2013-01-01 false Comparative evaluation of all sites proposed for nomination. 960.3-2-2-3 Section 960.3-2-2-3 Energy DEPARTMENT OF ENERGY GENERAL GUIDELINES FOR THE PRELIMINARY SCREENING OF POTENTIAL SITES FOR A NUCLEAR WASTE REPOSITORY Implementation Guidelines §...

  9. 10 CFR 960.3-2-2-3 - Comparative evaluation of all sites proposed for nomination.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 4 2014-01-01 2014-01-01 false Comparative evaluation of all sites proposed for nomination. 960.3-2-2-3 Section 960.3-2-2-3 Energy DEPARTMENT OF ENERGY GENERAL GUIDELINES FOR THE PRELIMINARY SCREENING OF POTENTIAL SITES FOR A NUCLEAR WASTE REPOSITORY Implementation Guidelines §...

  10. 10 CFR 960.3-2-2-3 - Comparative evaluation of all sites proposed for nomination.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Comparative evaluation of all sites proposed for nomination. 960.3-2-2-3 Section 960.3-2-2-3 Energy DEPARTMENT OF ENERGY GENERAL GUIDELINES FOR THE PRELIMINARY SCREENING OF POTENTIAL SITES FOR A NUCLEAR WASTE REPOSITORY Implementation Guidelines §...

  11. Are participant characteristics from ISCOLE study sites comparable to the rest of their country?

    PubMed Central

    LeBlanc, A G; Katzmarzyk, P T; Barreira, T V; Broyles, S T; Chaput, J-P; Church, T S; Fogelholm, M; Harrington, D M; Hu, G; Kuriyan, R; Kurpad, A; Lambert, E V; Maher, C; Maia, J; Matsudo, V; Olds, T; Onywera, V; Sarmiento, O L; Standage, M; Tudor-Locke, C; Zhao, P; Tremblay, M S

    2015-01-01

    OBJECTIVES: The International Study of Childhood Obesity, Lifestyle and the Environment (ISCOLE) provides robust, multi-national information on physical activity, diet and weight status in 9–11-year-old children around the world. The purpose of this analysis was to examine the similarities and differences between participant characteristics from ISCOLE sites and data from nationally representative surveys from ISCOLE countries (Australia, Brazil, Canada, China, Colombia, Finland, Kenya, India, Portugal, South Africa, the United Kingdom and the United States). METHODS: Distributions of characteristics were assessed within each ISCOLE country-level database, and compared with published data from national or regional surveys, where available. Variables of comparison were identified a priori and included body mass index (BMI), physical activity (accelerometer-determined steps per day) and screen time (child-report). RESULTS: Of 12 countries, data on weight status (BMI) were available in 8 countries, data on measured physical activity (steps per day) were available in 5 countries and data on self-reported screen time were available in 9 countries. The five ISCOLE countries that were part of the Health Behaviour in School-aged Children Survey (that is, Canada, Finland, Portugal, the United Kingdom (England) and the United States) also provided comparable data on self-reported physical activity. Available country-specific data often used different measurement tools or cut-points, making direct comparisons difficult. Where possible, ISCOLE data were re-analyzed to match country-level data, but this step limited between-country comparisons. CONCLUSIONS: From the analyses performed, the ISCOLE data do not seem to be systematically biased; however, owing to limitations in data availability, data from ISCOLE should be used with appropriate caution when planning country-level population health interventions. This work highlights the need for harmonized measurement tools

  12. Comparative assessment of five potential sites for hydrothermal-magma systems: energy transport

    SciTech Connect

    Hardee, H.C.

    1980-09-01

    A comparative assessment of five sites is being prepared as part of a Continental Scientific Drilling Program (CSDP) review of thermal regimes for the purpose of scoping areas for future research and drilling activities. This background report: discusses the various energy transport processes likely to be encountered in a hydrothermal-magma system, reviews related literature, discusses research and field data needs, and reviews the sites from an energy transport viewpoint. At least three major zones exist in the magma-hydrothermal transport system: the magma zone, the hydrothermal zone, and the transition zone between the two. Major energy transport questions relate to the nature and existence of these zones and their evolution with time. Additional energy transport questions are concerned with the possible existence of critical state and super-critical state permeable convection in deep geothermal systems. A review of thermal transport models emphasizes the fact that present transport models and computational techniques far outweigh the scarcity and quality of deep field data.

  13. Identification of putative active site residues of ACAT enzymes.

    PubMed

    Das, Akash; Davis, Matthew A; Rudel, Lawrence L

    2008-08-01

    In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

  14. 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

  15. 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.

  16. Proteome-wide analysis of nonsynonymous single-nucleotide variations in active sites of human proteins.

    PubMed

    Dingerdissen, Hayley; Motwani, Mona; Karagiannis, Konstantinos; Simonyan, Vahan; Mazumder, Raja

    2013-03-01

    An enzyme's active site is essential to normal protein activity such that any disruptions at this site may lead to dysfunction and disease. Nonsynonymous single-nucleotide variations (nsSNVs), which alter the amino acid sequence, are one type of disruption that can alter the active site. When this occurs, it is assumed that enzyme activity will vary because of the criticality of the site to normal protein function. We integrate nsSNV data and active site annotations from curated resources to identify all active-site-impacting nsSNVs in the human genome and search for all pathways observed to be associated with this data set to assess the likely consequences. We find that there are 934 unique nsSNVs that occur at the active sites of 559 proteins. Analysis of the nsSNV data shows an over-representation of arginine and an under-representation of cysteine, phenylalanine and tyrosine when comparing the list of nsSNV-impacted active site residues with the list of all possible proteomic active site residues, implying a potential bias for or against variation of these residues at the active site. Clustering analysis shows an abundance of hydrolases and transferases. Pathway and functional analysis shows several pathways over- or under-represented in the data set, with the most significantly affected pathways involved in carbohydrate metabolism. We provide a table of 32 variation-substrate/product pairs that can be used in targeted metabolomics experiments to assay the effects of specific variations. In addition, we report the significant prevalence of aspartic acid to histidine variation in eight proteins associated with nine diseases including glycogen storage diseases, lacrimo-auriculo-dento-digital syndrome, Parkinson's disease and several cancers.

  17. Donor-site Morbidity of Medial and Lateral Thigh-based Flaps: A Comparative Study

    PubMed Central

    Purnell, Chad A.; Lewis, Kevin C.; Mioton, Lauren M.; Hanwright, Philip J.; Galiano, Robert D.; Dumanian, Gregory A.; Alghoul, Mohammed S.

    2016-01-01

    Background: Free and pedicled medial and lateral thigh-based flaps are common reconstructive procedures. However, there have been no comparative studies of morbidity between medial and lateral donor sites. Methods: We conducted an Enterprise Data Warehouse-based review of all the senior authors’ (R.D.G., G.A.D., and M.S.A.) thigh-based free and pedicled flaps. Patient demographic data, donor-site complications, drain duration, and number of postoperative visits were collected and compared. Complications were also compared between fasciocutaneous flaps and muscle or myocutaneous flaps, and skin grafted donor sites. Results: We analyzed 352 flap donor sites, with 155 medial and 197 lateral. Two hundred seventeen (217) flaps were pedicled. Flap types included 127 gracilis, 27 rectus femoris, 134 anterolateral thigh, and 36 vastus lateralis-only flaps. There were no significant differences in complications between medial (17.4%) and lateral thigh (21.3%) donor sites, although lateral thigh flaps had a mean of 1 additional postoperative visit. Rates of wound dehiscence/healing issues were significantly higher in both gracilis myocutaneous flaps (25.9%) and flaps requiring a skin grafted donor site (31.2%). Postoperative therapeutic anticoagulation was the only significant risk factor for a donor-site complication. Flap complications resulted in increased drain duration and postoperative office visits. Conclusions: Donor-site morbidity is similar in both lateral and medial thigh-based flaps. The inclusion of muscle in the flap from either donor site does not seem to increase complications, but the inclusion of a skin paddle with gracilis muscle, or a skin grafted lateral thigh donor site, results in increased wound healing complications. PMID:27975004

  18. Barium ions selectively activate BK channels via the Ca2+-bowl site.

    PubMed

    Zhou, Yu; Zeng, Xu-Hui; Lingle, Christopher J

    2012-07-10

    Activation of Ca(2+)-dependent BK channels is increased via binding of micromolar Ca(2+) to two distinct high-affinity sites per BK α-subunit. One site, termed the Ca(2+) bowl, is embedded within the second RCK domain (RCK2; regulator of conductance for potassium) of each α-subunit, while oxygen-containing residues in the first RCK domain (RCK1) have been linked to a separate Ca(2+) ligation site. Although both sites are activated by Ca(2+) and Sr(2+), Cd(2+) selectively favors activation via the RCK1 site. Divalent cations of larger ionic radius than Sr(2+) are thought to be ineffective at activating BK channels. Here we show that Ba(2+), better known as a blocker of K(+) channels, activates BK channels and that this effect arises exclusively from binding at the Ca(2+)-bowl site. Compared with previous estimates for Ca(2+) bowl-mediated activation by Ca(2+), the affinity of Ba(2+) to the Ca(2+) bowl is reduced about fivefold, and coupling of binding to activation is reduced from ∼3.6 for Ca(2+) to about ∼2.8 for Ba(2+). These results support the idea that ionic radius is an important determinant of selectivity differences among different divalent cations observed for each Ca(2+)-binding site.

  19. Promoter-proximal polyadenylation sites reduce transcription activity

    PubMed Central

    Andersen, Pia K.; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels. PMID:23028143

  20. 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.

  1. 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.

  2. Active and regulatory sites of cytosolic 5'-nucleotidase.

    PubMed

    Pesi, Rossana; Allegrini, Simone; Careddu, Maria Giovanna; Filoni, Daniela Nicole; Camici, Marcella; Tozzi, Maria Grazia

    2010-12-01

    Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap₄A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap₄A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation.

  3. Comparative molecular modelling of biologically active sterols

    NASA Astrophysics Data System (ADS)

    Baran, Mariusz; Mazerski, Jan

    2015-04-01

    Membrane sterols are targets for a clinically important antifungal agent - amphotericin B. The relatively specific antifungal action of the drug is based on a stronger interaction of amphotericin B with fungal ergosterol than with mammalian cholesterol. Conformational space occupied by six sterols has been defined using the molecular dynamics method to establish if the conformational features correspond to the preferential interaction of amphotericin B with ergosterol as compared with cholesterol. The compounds studied were chosen on the basis of structural features characteristic for cholesterol and ergosterol and on available experimental data on the ability to form complexes with the antibiotic. Statistical analysis of the data obtained has been performed. The results show similarity of the conformational spaces occupied by all the sterols tested. This suggests that the conformational differences of sterol molecules are not the major feature responsible for the differential sterol - drug affinity.

  4. 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

  5. BAX Activation is Initiated at a Novel Interaction Site

    PubMed Central

    Gavathiotis, Evripidis; Suzuki, Motoshi; Davis, Marguerite L.; Pitter, Kenneth; Bird, Gregory H.; Katz, Samuel G.; Tu, Ho-Chou; Kim, Hyungjin; Cheng, Emily H.-Y.; Tjandra, Nico; Walensky, Loren D.

    2008-01-01

    BAX is a pro-apoptotic protein of the BCL-2 family stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed Stabilized Alpha-Helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the BIM SAHB-BAX interaction is highlighted by point mutagenesis that abrogates functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis. PMID:18948948

  6. Involvement of novel autophosphorylation sites in ATM activation.

    PubMed

    Kozlov, Sergei V; Graham, Mark E; Peng, Cheng; Chen, Philip; Robinson, Phillip J; Lavin, Martin F

    2006-08-09

    ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

  7. Single site and conventional totally extraperitoneal techniques for uncomplicated inguinal hernia repair: A comparative study

    PubMed Central

    de Araújo, Felipe Brandão Corrêa; Starling, Eduardo Simão; Maricevich, Marco; Tobias-Machado, Marcos

    2014-01-01

    OBJECTIVE: To demonstrate the feasibility of endoscopic extraperitoneal single site (EESS) inguinal hernia repair and compare it outcomes with the conventional totally extraperitoneal (TEP) technique. BACKGROUND: TEP inguinal hernia repair is a widely accepted alternative to conventional open technique with several perioperative advantages. Transumbilical laparoendoscopic singlesite surgery (LESS) is an emerging approach and has been reported for a number of surgical procedures with superior aesthetic results but other advantages need to be proven. PATIENTS AND METHODS: Thirty-eight uncomplicated inguinal hernias were repaired by EESS approach between January 2010 and January 2011. All procedures were performed through a 25 cm infraumbilical incision using the Alexis wound retractor attached to a surgical glove and three trocars. Body mass index, age, operative time, blood loss, complications, conversion rate, analgesia requirement, hospital stay, return to normal activities and patient satisfaction with aesthetic results were analysed and compared with the last 38 matched-pair group of patients who underwent a conventional TEP inguinal hernia repair by the same surgeon. RESULTS: All procedures were performed successfully with no conversion. In both unilateral and bilateral EESS inguinal repairs, the mean operative time was longer than conventional TEP (55± 20 vs. 40± 15 min, P = 0.049 and 70± 15 vs. 55± 10 min, P = 0.014). Aesthetic result was superior in the EESS group (2.88± 0.43 vs. 2.79± 0.51, P = 0.042). There was no difference between the two approaches regarding blood loss, complications, hospital stay, time until returns to normal activities and analgesic requirement. CONCLUSION: EESS inguinal hernia repair is safe and effective, with superior cosmetic results in the treatment of uncomplicated inguinal hernias. Other advantages of this new technique still need to be proven. PMID:25336820

  8. 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…

  9. Spectroscopic studies of the active site of galactose oxidase

    SciTech Connect

    Knowles, P.F.; Brown, R.D. III; Koenig, S.H.

    1995-07-19

    X-ray absorption and EPR spectroscopy have been used to probe the copper site structure in galactose oxidase at pH 4.5 and 7.0. the results suggest that there are no major differences in the structure of the tetragonal Cu(II) site at these pH values. Analysis of the extended X-ray absorption fine structure (EXAFS) indicates that four N,O scatterers are present at approximately 2 {Angstrom}; these are presumably the equatorial ligands. In addition, the EXAFS data establish that oxidative activation to produce the active-site tyrosine radical does not cause major changes in the copper coordination environment. Therefore results obtained on the one-electron reduced enzyme, containing Cu(II) but not the tyrosine radical, probably also apply to the catalytically active Cu(II)/tyrosine radical state. Solvent water exchange, inhibitor binding, and substrate binding have been probed via nuclear magnetic relaxation dispersion (NMRD) measurements. The NMRD profile of galactose oxidase is quantitatively consistent with the rapid exchange of a single, equatorial water ligand with a Cu(II)-O separation of about 2.4 {Angstrom}. Azide and cyanide displace this coordinated water. The binding of azide and the substrate dihydroxyacetone produce very similar effects on the NMRD profile of galactose oxidase, indicating that substrates also bind to the active site Cu(II) in an equatorial position.

  10. 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.

  11. Comparative modeling of an in situ diffusion experiment in granite at the Grimsel Test Site.

    PubMed

    Soler, Josep M; Landa, Jiri; Havlova, Vaclava; Tachi, Yukio; Ebina, Takanori; Sardini, Paul; Siitari-Kauppi, Marja; Eikenberg, Jost; Martin, Andrew J

    2015-08-01

    An in situ diffusion experiment was performed at the Grimsel Test Site (Switzerland). Several tracers ((3)H as HTO, (22)Na(+), (134)Cs(+), (131)I(-) with stable I(-) as carrier) were continuously circulated through a packed-off borehole and the decrease in tracer concentrations in the liquid phase was monitored for a period of about 2years. Subsequently, the borehole section was overcored and the tracer profiles in the rock analyzed ((3)H, (22)Na(+), (134)Cs(+)). (3)H and (22)Na(+) showed a similar decrease in activity in the circulation system (slightly larger drop for (3)H). The drop in activity for (134)Cs(+) was much more pronounced. Transport distances in the rock were about 20cm for (3)H, 10cm for (22)Na(+), and 1cm for (134)Cs(+). The dataset (except for (131)I(-) because of complete decay at the end of the experiment) was analyzed with different diffusion-sorption models by different teams (IDAEA-CSIC, UJV-Rez, JAEA) using different codes, with the goal of obtaining effective diffusion coefficients (De) and porosity (ϕ) or rock capacity (α) values. From the activity measurements in the rock, it was observed that it was not possible to recover the full tracer activity in the rock (no activity balance when adding the activities in the rock and in the fluid circulation system). A Borehole Disturbed Zone (BDZ) had to be taken into account to fit the experimental observations. The extension of the BDZ (1-2mm) is about the same magnitude than the mean grain size of the quartz and feldspar grains. IDAEA-CSIC and UJV-Rez tried directly to match the results of the in situ experiment, without forcing any laboratory-based parameter values into the models. JAEA conducted a predictive modeling based on laboratory diffusion data and their scaling to in situ conditions. The results from the different codes have been compared, also with results from small-scale laboratory experiments. Outstanding issues to be resolved are the need for a very large capacity factor in the

  12. 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.

  13. 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

    2016-09-06

    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.

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

    PubMed Central

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

    2016-01-01

    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

  15. Cellular Active N-Hydroxyurea FEN1 Inhibitors Block Substrate Entry to the Active Site

    PubMed Central

    Exell, Jack C.; Thompson, Mark J.; Finger, L. David; Shaw, Steven J.; Debreczeni, Judit; Ward, Thomas A.; McWhirter, Claire; Siöberg, Catrine L. B.; Martinez Molina, Daniel; Mark Abbott, W.; Jones, Clifford D.; Nissink, J. Willem M.; Durant, Stephen T.; Grasby, Jane A.

    2016-01-01

    The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the first crystal structure of inhibitor-bound hFEN1 and show a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein–substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the unpairing of substrate DNA necessary for reaction. Other compounds were more competitive with substrate. Cellular thermal shift data showed engagement of both inhibitor types with hFEN1 in cells with activation of the DNA damage response evident upon treatment. However, cellular EC50s were significantly higher than in vitro inhibition constants and the implications of this for exploitation of hFEN1 as a drug target are discussed. PMID:27526030

  16. The active site structure and mechanism of phosphoenolpyruvate utilizing enzymes

    SciTech Connect

    Cheng, K.C.

    1989-01-01

    Arginine specific reagents showed irreversible inhibition of avian liver mitochondrial phosphoenolpyruvate carboxykinase. Potent protection against modification was elicited by CO{sub 2} or CO{sub 2} in the presence of other substrates. Labeling of enzyme with (7-{sup 14}C) phenylglyoxal showed that 1 or 2 arginines are involved in CO{sub 2} binding and activation. Peptide map studies showed this active site arginine residues is located at position 289. Histidine specific reagents showed pseudo first order inhibition of avian mitochondrial phosphoenolpyruvate carboxykinase activity. The best protection against modification was elicited by IDP or IDP and Mn{sup +2}. One histidine residue is at or near the phosphoenolpyruvate binding site as demonstrated in the increased absorbance at 240 nm and proton relaxation rate studies. Circular dichroism studies reveal that enzyme structure was perturbed by diethylpyrocarbonate modification. Metal binding studies suggest that this enzyme has only one metal binding site. The putative binding sites from several GTP and phosphoenolpyruvate utilizing enzymes are observed in P-enolpyruvate carboxykinase from different species.

  17. SENSITIVITY ANALYSIS OF THE APPLICATION OF CHEMICAL EXPOSURE CRITERIA FOR COMPARING SITES AND WATERSHEDS

    EPA Science Inventory

    A methodology was developed for deriving quantitative exposure criteria useful for comparing a site or watershed to a reference condition. The prototype method used indicators of exposures to oil contamination and combustion by-products, naphthalene and benzo(a)pyrene metabolites...

  18. Nursing Home Administrators' Opinions of the Nursing Home Compare Web Site

    ERIC Educational Resources Information Center

    Castle, Nicholas G.

    2005-01-01

    Purpose: In November of 2002 the Centers for Medicare and Medicaid Services publicly reported on a national basis the quality of nursing homes on the Nursing Home Compare (NHC) Web site. This study examines administrators' opinions of this initiative and whether it has fostered quality improvement. Design and Methods: Data used in this…

  19. Comparative receptor mapping of serotoninergic 5-HT3 and 5-HT4 binding sites*

    NASA Astrophysics Data System (ADS)

    López-Rodríguez, María L.; Morcillo, María José; Benhamú, Bellinda; Rosado, María Luisa

    1997-11-01

    .8.0 Å from the aromatic moiety in the 5-HT4 antagonistpharmacophore, whereas this distance is ca. 7.5 Å in the5-HT3 antagonist model. The comparative mapping of bothserotoninergic receptors has allowed us to confirm the three-componentpharmacophore accepted for the 5-HT3 receptor, as well as topropose a steric model for the 5-HT4 receptor binding site. Thisstudy offers structural insights to aid the design of new selective ligands,and the resulting models have received some support from the synthesis of twonew active and selective ligands: 24 (Ki(5-HT3)= 3.7 nM; Ki(5-HT4) > 1000 nM) and 25(Ki(5-HT4) = 13.7 nM;Ki(5-HT3) > 10 000 nM).

  20. Comparative microhabitat characteristics at oviposition sites of the California red-legged frog (Rana draytonii)

    USGS Publications Warehouse

    Alvarez, Jeff A.; Cook, David G.; Yee, Julie L.; van Hattem, Michael G.; Fong, Darren R.; Fisher, Robert N.

    2013-01-01

    We studied the microhabitat characteristics of 747 egg masses of the federally-threatened Rana draytonii (California red-legged frog) at eight sites in California. our study showed that a broad range of aquatic habitats are utilized by ovipositing R. draytonii, including sites with perennial and ephemeral water sources, natural and constructed wetlands, lentic and lotic hydrology, and sites surrounded by protected lands and nested within modified urban areas. We recorded 45 different egg mass attachment types, although the use of only a few types was common at each site. These attachment types ranged from branches and roots of riparian trees, emergent and submergent wetland vegetation, flooded upland grassland/ruderal vegetation, and debris. eggs were deposited in relatively shallow water (mean 39.7 cm) when compared to maximum site depths. We found that most frogs in artificial pond, natural creek, and artificial channel habitats deposited egg masses within one meter of the shore, while egg masses in a seasonal marsh averaged 27.3 m from the shore due to extensive emergent vegetation. Rana draytonii appeared to delay breeding in lotic habitats and in more inland sites compared to lentic habitats and coastal sites. eggs occurred as early as mid-december at a coastal artificial pond and as late as mid-April in an inland natural creek. We speculate that this delay in breeding may represent a method of avoiding high-flow events and/or freezing temperatures. Understanding the factors related to the reproductive needs of this species can contribute to creating, managing, or preserving appropriate habitat, and promoting species recovery.

  1. Specific mixing facilitates the comparative quantification of phosphorylation sites with significant dysregulations.

    PubMed

    Liu, Jing; Xu, Bo; Liu, Zheyi; Dong, Mingming; Mao, Jiawei; Zhou, Ye; Chen, Jin; Wang, Fangjun; Zou, Hanfa

    2017-01-15

    Mass spectrometry (MS) based quantitative analyses of proteome and proteome post-translational modifications (PTMs) play more and more important roles in biological, pharmaceutical and clinical studies. However, it is still a big challenge to accurately quantify the proteins or proteins PTM sites with extreme relative abundances in comparative protein samples, such as the significantly dysregulated ones. Herein, a novel quantification strategy, Mixing at Specific Ratio (MaSR) before isotope labeling, had been developed to improve the quantification accuracy and coverage of extreme proteins and protein phosphorylation sites. Briefly, the comparative protein samples were firstly mixed together at specific ratios of 9:1 and 1:9 (w/w), followed with mass differentiate light and heavy isotope labeling, respectively. The extreme proteins and protein phosphorylation sites, even if the newly expressed or disappeared ones, could be accurately quantified due to all of the proteins' relative abundances had been adjusted to 2 orders of magnitude (1/9-9) by this strategy. The number of quantified phosphorylation sites with more than 20 folds changes was improved about 10 times in comparative quantification of pervanadate stimulated phosphoproteome of HeLa cells, and 134 newly generated and 21 disappeared phosphorylation sites were solely quantified by the MaSR strategy. The significantly up-regulated phosphorylation sites were mainly involved in the key phosphoproteins regulating the insulin-related pathways, such as PI3K-AKT and RAS-MAPK pathways. Therefore, the MaSR strategy exhibits as a promising way in elucidating the biological processes with significant dysregulations.

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  3. Comparative assessment of five potential sites for magma: hydrothermal systems - geophysics

    SciTech Connect

    Kasameyer, P.

    1980-09-02

    As part of a comparative assessment for the Continental Scientific Drilling Program, geophysical data were used, to characterize and evaluate potential magma-hydrothermal targets at five drill sites in the western United States. The sites include Roosevelt Hot Springs, Utah, the Rio Grande Rift, New Mexico, and The Geysers-Clear Lake, Long Valley, and Salton Trough areas, California. This summary discusses the size, depth, temperature, and setting of each potential target, as well as relvant scientific questions about their natures and the certainty of their existence.

  4. Comparing the Chemistry for the Pathfinder, MER and MSL Martian Landing Sites with APXS

    NASA Astrophysics Data System (ADS)

    Gellert, R.; Arvidson, R. E.; Clark, B. C.; Grotzinger, J. P.; Squyres, S. W.; Yen, A. S.

    2013-12-01

    The Alpha-Particle-X-ray Spectrometer is part of the science payload of all recent NASA Mars rover missions. It allows detailed comparison of the chemical composition and overall findings from rocks and soils at 4 landing sites. It can be even extended to the soil measurements performed with XRF on the two Viking landers. Typical soils at all 6 landing sites are very similar in chemical composition as well as mineralogy from instruments like Moessbauer on MER and CHEMIN on MSL. Beside a dominant basaltic composition the soil contains a significant altered and volatile rich component that is characterized by a constant S/Cl ratio in the APXS data. The overall similarity of the soils as well as the finding that soils around the 30 wt % SO3 rich bedrocks at Meridiani are not elevated in S, indicates the presence of a global distributed or at least large scale homogenized component in the Martian soil. This allows extending the SAM and CHEMIN findings in the altered and volatile components in the Gale Rocknest soils to other landing sites. However, in Gusev Crater anomalous subsurface soils were found - silica and hydrated ferric sulfate rich - that could indicate local fumarole and leaching activities. The sulfate rich Meridiani bedrock, called Burns formations, was documented to be remarkably homogeneous over the ~ 15 miles from the landing site at Eagle Crater to the base of the rim at Endurance Crater. Here for the first time a rover entered an area where evidence for clays is given by orbital CRISM data. With Opportunity's mineralogy instruments out of commission, the APXS was used to document the chemistry of the encountered outcrops. Having a composition close to the average Mars, one main characteristic feature of rocks at the rim is the low sulfur content compared to the younger Meridiani bedrock. The outcrops show post depositional cracks and veins filled with Calciumsulfate and Chlorine rich surface alteration rinds. A smaller outcrop called Esperance has

  5. 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.

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

    PubMed Central

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

    2000-01-01

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

  7. Active site amino acid sequence of human factor D.

    PubMed

    Davis, A E

    1980-08-01

    Factor D was isolated from human plasma by chromatography on CM-Sephadex C50, Sephadex G-75, and hydroxylapatite. Digestion of reduced, S-carboxymethylated factor D with cyanogen bromide resulted in three peptides which were isolated by chromatography on Sephadex G-75 (superfine) equilibrated in 20% formic acid. NH2-Terminal sequences were determined by automated Edman degradation with a Beckman 890C sequencer using a 0.1 M Quadrol program. The smallest peptide (CNBr III) consisted of the NH2-terminal 14 amino acids. The other two peptides had molecular weights of 17,000 (CNBr I) and 7000 (CNBr II). Overlap of the NH2-terminal sequence of factor D with the NH2-terminal sequence of CNBr I established the order of the peptides. The NH2-terminal 53 residues of factor D are somewhat more homologous with the group-specific protease of rat intestine than with other serine proteases. The NH2-terminal sequence of CNBr II revealed the active site serine of factor D. The typical serine protease active site sequence (Gly-Asp-Ser-Gly-Gly-Pro was found at residues 12-17. The region surrounding the active site serine does not appear to be more highly homologous with any one of the other serine proteases. The structural data obtained point out the similarities between factor D and the other proteases. However, complete definition of the degree of relationship between factor D and other proteases will require determination of the remainder of the primary structure.

  8. 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.

  9. [Mechanism of arginine deiminase activity by site-directed mutagenesis].

    PubMed

    Li, Lifeng; Ni, Ye; Sun, Zhihao

    2012-04-01

    Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

  10. Potential sites of CFTR activation by tyrosine kinases

    PubMed Central

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

    2016-01-01

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

  11. Northern Idaho house dust and soil lead levels compared to the Bunker Hill Superfund Site.

    PubMed

    Spalinger, Susan M; von Braun, Margrit C; Petrosyan, Varduhi; von Lindern, Ian H

    2007-07-01

    House dust has been identified as a major exposure medium for lead (Pb) in children. High levels of Pb in soil and house dust have been recorded at the Bunker Hill Superfund Site (BHSS) in northern Idaho, an historic mining and smelting district. Soil and dust remediation at the site was required; however, regional background soil and dust Pb levels had not been well characterized. The objective of this survey was to determine background house dust Pb levels and to compare those levels with concentrations, and dust and Pb loading rates measured at the BHSS. Soil and house dust samples were collected in five towns demographically similar to the BHSS but unaffected by the mining industry. The background concentrations and loading rates were significantly lower than those observed at the site. House age was a significant factor affecting background soil and house dust Pb concentrations and loading rates.

  12. MSK1 activity is controlled by multiple phosphorylation sites

    PubMed Central

    McCOY, Claire E.; Campbell, David G.; Deak, Maria; Bloomberg, Graham B.; Arthur, J. Simon C.

    2004-01-01

    MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2. PMID:15568999

  13. Cold-active enzymes studied by comparative molecular dynamics simulation.

    PubMed

    Spiwok, Vojtech; Lipovová, Petra; Skálová, Tereza; Dusková, Jarmila; Dohnálek, Jan; Hasek, Jindrich; Russell, Nicholas J; Králová, Blanka

    2007-04-01

    Enzymes from cold-adapted species are significantly more active at low temperatures, even those close to zero Celsius, but the rationale of this adaptation is complex and relatively poorly understood. It is commonly stated that there is a relationship between the flexibility of an enzyme and its catalytic activity at low temperature. This paper gives the results of a study using molecular dynamics simulations performed for five pairs of enzymes, each pair comprising a cold-active enzyme plus its mesophilic or thermophilic counterpart. The enzyme pairs included alpha-amylase, citrate synthase, malate dehydrogenase, alkaline protease and xylanase. Numerous sites with elevated flexibility were observed in all enzymes; however, differences in flexibilities were not striking. Nevertheless, amino acid residues common in both enzymes of a pair (not present in insertions of a structure alignment) are generally more flexible in the cold-active enzymes. The further application of principle component analysis to the protein dynamics revealed that there are differences in the rate and/or extent of opening and closing of the active sites. The results indicate that protein dynamics play an important role in catalytic processes where structural rearrangements, such as those required for active site access by substrate, are involved. They also support the notion that cold adaptation may have evolved by selective changes in regions of enzyme structure rather than in global change to the whole protein.

  14. 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.

  15. Vitamin K epoxide reductase: homology, active site and catalytic mechanism.

    PubMed

    Goodstadt, Leo; Ponting, Chris P

    2004-06-01

    Vitamin K epoxide reductase (VKOR) recycles reduced vitamin K, which is used subsequently as a co-factor in the gamma-carboxylation of glutamic acid residues in blood coagulation enzymes. VKORC1, a subunit of the VKOR complex, has recently been shown to possess this activity. Here, we show that VKORC1 is a member of a large family of predicted enzymes that are present in vertebrates, Drosophila, plants, bacteria and archaea. Four cysteine residues and one residue, which is either serine or threonine, are identified as likely active-site residues. In some plant and bacterial homologues the VKORC1 homologous domain is fused with domains of the thioredoxin family of oxidoreductases. These might reduce disulfide bonds of VKORC1-like enzymes as a prerequisite for their catalytic activities.

  16. Substrate shuttling between active sites of uroporphyrinogen decarboxylase is not required to generate coproporphyrinogen

    PubMed Central

    Phillips, John D.; Warby, Christy A.; Whitby, Frank G.; Kushner, James P.; Hill, Christopher P.

    2009-01-01

    Summary 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 the four acetate side chains on 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 (scURO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposible with wild-type activity and have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of scURO-D resulted in approximately half of wild-type activity. The distribution of reaction intermediates was the same for mutant and wild-type sequences, and was unaltered in a competition experiment using the 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 create a large active site cleft. PMID:19362562

  17. 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.

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

    PubMed Central

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

    2015-01-01

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

  19. 75 FR 58411 - Medicare Program; Town Hall Meeting on the Physician Compare Web Site, October 27, 2010

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-24

    ... Physician Compare Web Site, October 27, 2010 AGENCY: Centers for Medicare & Medicaid Services (CMS), HHS... 2010, ``Public Reporting of Performance Information'' requires CMS to establish a Physician Compare Web site by January 1, 2011. This notice announces a Town Hall meeting to discuss the Physician Compare...

  20. Comparative analysis of effluent water quality from a municipal treatment plant and two on-site wastewater treatment systems.

    PubMed

    Garcia, Santos N; Clubbs, Rebekah L; Stanley, Jacob K; Scheffe, Brian; Yelderman, Joe C; Brooks, Bryan W

    2013-06-01

    Though decentralized on-site technologies are extensively employed for wastewater treatment around the globe, an understanding of effluent water quality impairments associated with these systems remain less understood than effluent discharges from centralized municipal wastewater treatment facilities. Using a unique experimental facility, a novel comparative analysis of effluent water quality was performed from model decentralized aerobic (ATS) and septic (STS) on-site wastewater treatment systems and a centralized municipal wastewater treatment plant (MTP). The ATS and STS units did not benefit from further soil treatment. Each system received common influent wastewater from the Waco, Texas, USA Metropolitan Area Regional Sewerage System. We tested the hypothesis that MTP effluent would exhibit higher water quality than on-site effluents, based on parameters selected for study. A tiered testing approach was employed to assess the three effluent discharges: select routine water quality parameters (Tier I), whole effluent toxicity (Tier II), and select endocrine-active compounds (Tier III). Contrary to our hypothesis, ATS effluent was not statistically different from MTP effluents, based on Tier I and III parameters, but reproductive responses of Daphnia magna were slightly more sensitive to ATS than MTP effluents. STS effluent water quality was identified as most degraded of the three wastewater treatment systems. Parameters used to assess centralized wastewater treatment plant effluent water quality such as whole effluent toxicity and endocrine active substances appear useful for water quality assessments of decentralized discharges. Aerobic on-site wastewater treatment systems may represent more robust options than traditional septic systems for on-site wastewater treatment in watersheds with appreciable groundwater - surface water exchange.

  1. 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.

  2. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  3. Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity.

    PubMed

    Eaves, Deborah J; Haque, Tamanna; Tudor, Richard L; Barron, Yoshimi; Zampronio, Cleidiane G; Cotton, Nicholas P J; de Graaf, Barend H J; White, Scott A; Cooper, Helen J; Franklin, F Christopher H; Harper, Jeffery F; Franklin-Tong, Vernonica E

    2017-03-01

    Protein phosphorylation regulates numerous cellular processes. Identifying the substrates and protein kinases involved is vital to understand how these important posttranslational modifications modulate biological function in eukaryotic cells. Pyrophosphatases catalyze the hydrolysis of inorganic phosphate (PPi) to inorganic phosphate Pi, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate. It was suggested recently that posttranslational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. We previously demonstrated that two pollen-expressed sPPases, Pr-p26.1a and Pr-p26.1b, from the flowering plant Papaver rhoeas were inhibited by phosphorylation. Despite the potential significance, there is a paucity of data on sPPase phosphorylation and regulation. Here, we used liquid chromatographic tandem mass spectrometry to map phosphorylation sites to the otherwise divergent amino-terminal extensions on these pollen sPPases. Despite the absence of reports in the literature on mapping phosphorylation sites on sPPases, a database survey of various proteomes identified a number of examples, suggesting that phosphorylation may be a more widely used mechanism to regulate these enzymes. Phosphomimetic mutants of Pr-p26.1a/b significantly and differentially reduced PPase activities by up to 2.5-fold at pH 6.8 and 52% in the presence of Ca(2+) and hydrogen peroxide over unmodified proteins. This indicates that phosphoregulation of key sites can inhibit the catalytic responsiveness of these proteins in concert with key intracellular events. As sPPases are essential for many metabolic pathways in eukaryotic cells, our findings identify the phosphorylation of sPPases as a potential master regulatory mechanism that could be used to attenuate metabolism.

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

    SciTech Connect

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

    1986-05-01

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

  5. First Principles Computational Study of the Active Site of Arginase

    SciTech Connect

    Ivanov, Ivaylo; Klien, Micheal

    2004-01-14

    Ab initio density functional theory (DFT) methods were used to investigate the structural features of the active site of the binuclear enzyme rat liver arginase. Special emphasis was placed on the crucial role of the second shell ligand interactions. These interactions were systematically studied by performing calculations on models of varying size. It was determined that a water molecule, and not hydroxide, is the bridging exogenous ligand. The carboxylate ligands facilitate the close approach of the Mn (II) ions by attenuating the metal-metal electrostatic repulsion. Of the two metals, MnA was shown to carry a larger positive charge. Analysis of the electronic properties of the active site revealed that orbitals involving the terminal Asp234 residue, as well as the flexible -1,1 bridging Asp232, lie at high energies, suggesting weaker coordination. This is reflected in certain structural variability present in our models and is also consistent with recent experimental findings. Finally, implications of our findings for the biological function of the enzyme are delineated.

  6. Physical activity changes during pregnancy in a comparative impact trail

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Delta Healthy Sprouts was designed to test the comparative impact of two home visiting programs on weight status, dietary intake, physical activity, and other health behaviors of rural, Southern African American women and their infants. Results pertaining to physical activity outcomes in the gestat...

  7. C-H Activation on Co,O Sites: Isolated Surface Sites versus Molecular Analogs.

    PubMed

    Estes, Deven P; Siddiqi, Georges; Allouche, Florian; Kovtunov, Kirill V; Safonova, Olga V; Trigub, Alexander L; Koptyug, Igor V; Copéret, Christophe

    2016-11-16

    The activation and conversion of hydrocarbons is one of the most important challenges in chemistry. Transition-metal ions (V, Cr, Fe, Co, etc.) isolated on silica surfaces are known to catalyze such processes. The mechanisms of these processes are currently unknown but are thought to involve C-H activation as the rate-determining step. Here, we synthesize well-defined Co(II) ions on a silica surface using a metal siloxide precursor followed by thermal treatment under vacuum at 500 °C. We show that these isolated Co(II) sites are catalysts for a number of hydrocarbon conversion reactions, such as the dehydrogenation of propane, the hydrogenation of propene, and the trimerization of terminal alkynes. We then investigate the mechanisms of these processes using kinetics, kinetic isotope effects, isotopic labeling experiments, parahydrogen induced polarization (PHIP) NMR, and comparison with a molecular analog. The data are consistent with all of these reactions occurring by a common mechanism, involving heterolytic C-H or H-H activation via a 1,2 addition across a Co-O bond.

  8. 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

  9. Comparative simulation studies of native and single-site mutant human beta-defensin-1 peptides.

    PubMed

    Toubar, Rabab A; Zhmurov, Artem; Barsegov, Valeri; Marx, Kenneth A

    2013-01-01

    Human defensins play important roles in a broad range of biological functions, such as microbial defense and immunity. Yet, little is known about their molecular properties, i.e. secondary structure stability, structural variability, important side chain interactions, surface charge distribution, and resistance to thermal fluctuations, and how these properties are related to their functions. To assess these factors, we studied the native human β-defensin-1 monomer and dimer as well as several single-site mutants using molecular dynamics simulations. The results showed that disulfide bonds are important determinants in maintaining the defensins' structural integrity, as no structural transitions were observed at 300 K and only minor structural unfolding was detected upon heating to 500 K. The α-helix was less thermally stable than the core β-sheet structure held together by hydrogen bonds and hydrophobic interactions. The monomer α-helix stability was directly correlated, whereas the end-to-end distance was inversely correlated to the experimentally measured β-defensin-1 chemotactic activity, in the order: mutant 2 (Gln24Glu) > mutant 3 (Lys31Ala) = wild type > mutant 1 (Asn4Ala). The structural stability of the β-defensin-1 dimer species exhibited an inverse correlation to their chemotactic activity. In dimers formed by mutants 2 and 3, we observed sliding of one monomer upon the surface of the other in the absence of unbinding. This dynamic sliding feature may enhance the molecular oligomerization of β-defensin-1 peptides contributing to their antibacterial activity. It could also help these peptides orient correctly in the CC chemokine receptor 6 binding site, thereby initiating their chemotactic activity. In agreement with this notion, the remarkable sliding behavior was observed only for the mutants with the highest chemotactic activity.

  10. Structure and nuclearity of active sites in Fe-zeolites: comparison with iron sites in enzymes and homogeneous catalysts.

    PubMed

    Zecchina, Adriano; Rivallan, Mickaël; Berlier, Gloria; Lamberti, Carlo; Ricchiardi, Gabriele

    2007-07-21

    Fe-ZSM-5 and Fe-silicalite zeolites efficiently catalyse several oxidation reactions which find close analogues in the oxidation reactions catalyzed by homogeneous and enzymatic compounds. The iron centres are highly dispersed in the crystalline matrix and on highly diluted samples, mononuclear and dinuclear structures are expected to become predominant. The crystalline and robust character of the MFI framework has allowed to hypothesize that the catalytic sites are located in well defined crystallographic positions. For this reason these catalysts have been considered as the closest and best defined heterogeneous counterparts of heme and non heme iron complexes and of Fenton type Fe(2+) homogeneous counterparts. On this basis, an analogy with the methane monooxygenase has been advanced several times. In this review we have examined the abundant literature on the subject and summarized the most widely accepted views on the structure, nuclearity and catalytic activity of the iron species. By comparing the results obtained with the various characterization techniques, we conclude that Fe-ZSM-5 and Fe-silicalite are not the ideal samples conceived before and that many types of species are present, some active and some other silent from adsorptive and catalytic point of view. The relative concentration of these species changes with thermal treatments, preparation procedures and loading. Only at lowest loadings the catalytically active species become the dominant fraction of the iron species. On the basis of the spectroscopic titration of the active sites by using NO as a probe, we conclude that the active species on very diluted samples are isolated and highly coordinatively unsaturated Fe(2+) grafted to the crystalline matrix. Indication of the constant presence of a smaller fraction of Fe(2+) presumably located on small clusters is also obtained. The nitrosyl species formed upon dosing NO from the gas phase on activated Fe-ZSM-5 and Fe-silicalite, have been analyzed

  11. 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.

  12. Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus kandleri.

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

    Aminoacyl tRNA synthetases (aaRSs) carry out the first step of protein biosynthesis. Several aaRSs are multimeric, and coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and accurate aminoacylation. However, important lacunae of understanding exist concerning the conformational dynamics of multimeric aaRSs. Questions remained unanswered pertaining to the dynamics of the active site. Little is known concerning the conformational dynamics of the active sites in response to the substrate binding, reorganization of the catalytic residues around reactants, time-dependent changes at the reaction center, which are essential for facilitating the nucleophilic attack, and interactions at the interface of neighboring monomers. In the present work, we carried out all-atom molecular dynamics simulation of dimeric (mk)SerRS from Methanopyrus kandleri bound with tRNA using an explicit solvent system. Two dimeric states of seryl tRNA synthetase (open, substrate bound, and adenylate bound) and two monomeric states (open and substrate bound) are simulated with bound tRNA. The aim is to understand the conformational dynamics of (mk)SerRS during its reaction cycle. While the present results provide a clear dynamical perspective of the active sites of (mk)SerRS, they corroborate with the results from the time-averaged experimental data such as crystallographic and mutation analysis of methanogenic SerRS from M. kandleri and M. barkeri. It is observed from the present simulation that the motif 2 loop gates the active site and its Glu351 and Arg360 stabilizes ATP in a bent state favorable for nucleophilic attack. The flexibility of the walls of the active site gradually reduces near reaction center, which is a more organized region compared to the lid region. The motif 2 loop anchors Ser and ATP using Arg349 in a hydrogen bonded geometry crucial for nucleophilic attack and favorably influences the electrostatic potential at the

  13. Stormwater management criteria for on-site pollution control: a comparative assessment of international practices.

    PubMed

    Sage, Jérémie; Berthier, Emmanuel; Gromaire, Marie-Christine

    2015-07-01

    Over the last decade, a growing interest has been shown toward innovative stormwater management practices, breaking away from conventional "end of pipe" approaches (based on conveying water offsite to centralized detention facilities). Innovative strategies, referred to as sustainable urban drainage systems, low impact development (LID) or green infrastructures, advocating for management of runoff as close to its origin as possible, have therefore gained a lot of popularity among practitioners and public authorities. However, while the need for pollution control is generally well accepted, there is no wide agreement about management criteria to be given to developers. This article hence aims to compare these criteria through literature analysis of different state or local stormwater management manuals or guidelines, investigating both their suitability for pollution control and their influence on best management practices selection and design. Four categories of criteria were identified: flow-rate limitations, "water quality volumes" (to be treated), volume reduction (through infiltration or evapotranspiration), and non-hydrologic criteria (such as loads reduction targets or maximum effluent concentrations). This study suggests that hydrologic criteria based on volume reduction (rather than treatment) might generally be preferable for on-site control of diffuse stormwater pollution. Nonetheless, determination of an appropriate management approach for a specific site is generally not straightforward and presents a variety of challenges for site designers seeking to satisfy local requirements in addressing stormwater quantity and quality issues. The adoption of efficient LID solution may therefore strongly depend on the guidance given to practitioners to account for these management criteria.

  14. Stormwater Management Criteria for On-Site Pollution Control: A Comparative Assessment of International Practices

    NASA Astrophysics Data System (ADS)

    Sage, Jérémie; Berthier, Emmanuel; Gromaire, Marie-Christine

    2015-07-01

    Over the last decade, a growing interest has been shown toward innovative stormwater management practices, breaking away from conventional "end of pipe" approaches (based on conveying water offsite to centralized detention facilities). Innovative strategies, referred to as sustainable urban drainage systems, low impact development (LID) or green infrastructures, advocating for management of runoff as close to its origin as possible, have therefore gained a lot of popularity among practitioners and public authorities. However, while the need for pollution control is generally well accepted, there is no wide agreement about management criteria to be given to developers. This article hence aims to compare these criteria through literature analysis of different state or local stormwater management manuals or guidelines, investigating both their suitability for pollution control and their influence on best management practices selection and design. Four categories of criteria were identified: flow-rate limitations, "water quality volumes" (to be treated), volume reduction (through infiltration or evapotranspiration), and non-hydrologic criteria (such as loads reduction targets or maximum effluent concentrations). This study suggests that hydrologic criteria based on volume reduction (rather than treatment) might generally be preferable for on-site control of diffuse stormwater pollution. Nonetheless, determination of an appropriate management approach for a specific site is generally not straightforward and presents a variety of challenges for site designers seeking to satisfy local requirements in addressing stormwater quantity and quality issues. The adoption of efficient LID solution may therefore strongly depend on the guidance given to practitioners to account for these management criteria.

  15. Comparing the Scoring of Human Decomposition from Digital Images to Scoring Using On-site Observations.

    PubMed

    Dabbs, Gretchen R; Bytheway, Joan A; Connor, Melissa

    2017-01-25

    When in forensic casework or empirical research in-person assessment of human decomposition is not possible, the sensible substitution is color photographic images. To date, no research has confirmed the utility of color photographic images as a proxy for in situ observation of the level of decomposition. Sixteen observers scored photographs of 13 human cadavers in varying decomposition stages (PMI 2-186 days) using the Total Body Score system (total n = 929 observations). The on-site TBS was compared with recorded observations from digital color images using a paired samples t-test. The average difference between on-site and photographic observations was -0.20 (t = -1.679, df = 928, p = 0.094). Individually, only two observers, both students with <1 year of experience, demonstrated TBS statistically significantly different than the on-site value, suggesting that with experience, observations of human decomposition based on digital images can be substituted for assessments based on observation of the corpse in situ, when necessary.

  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. 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.

  18. 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.

  19. An active site water network in the plasminogen activator pla from Yersinia pestis.

    PubMed

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

    2010-07-14

    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 A. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  20. Comparative toxicology of laboratory organisms for assessing hazardous-waste sites

    SciTech Connect

    Miller, W.E.; Peterson, S.A.; Greene, J.C.; Callahan, C.A.

    1985-01-01

    Multi-media/multi-trophic level bioassays have been proposed to determine the extent and severity of environmental contamination at hazardous waste sites. Comparative toxicological profiles for algae, daphnia, earthworms, microbes, mixed sewage and plants; wheat Stephens, lettuce, butter crunch, radish, Cherry Belle, red clover, Kenland, and cucumber, Spartan Valor are presented for selected heavy metals, herbicides and insecticides. Specific chemical EC50 values are presented for each test organism. Differences in standard deviations were compared between each individual test organism, as well as for the chemical subgroup assayed. Algae and daphnia are the most sensitive test organisms to heavy metals and insecticides followed in order of decreasing sensitivity by Microtox, DO depletion rate, seed germination and earthworms. Differences in toxicity of 2,4-D chemical formulations and commercial sources of insecticides were observed with algae and daphnia tests.

  1. 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

  2. Comparing the landcapes of common retroviral insertion sites across tumor models

    NASA Astrophysics Data System (ADS)

    Weishaupt, Holger; Čančer, Matko; Engström, Cristopher; Silvestrov, Sergei; Swartling, Fredrik J.

    2017-01-01

    Retroviral tagging represents an important technique, which allows researchers to screen for candidate cancer genes. The technique is based on the integration of retroviral sequences into the genome of a host organism, which might then lead to the artificial inhibition or expression of proximal genetic elements. The identification of potential cancer genes in this framework involves the detection of genomic regions (common insertion sites; CIS) which contain a number of such viral integration sites that is greater than expected by chance. During the last two decades, a number of different methods have been discussed for the identification of such loci and the respective techniques have been applied to a variety of different retroviruses and/or tumor models. We have previously established a retrovirus driven brain tumor model and reported the CISs which were found based on a Monte Carlo statistics derived detection paradigm. In this study, we consider a recently proposed alternative graph theory based method for identifying CISs and compare the resulting CIS landscape in our brain tumor dataset to those obtained when using the Monte Carlo approach. Finally, we also employ the graph-based method to compare the CIS landscape in our brain tumor model with those of other published retroviral tumor models.

  3. 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

    Highlights: •The disruption of PREG/PROG hydroxylation activity by T306A showed the participation of Cpd I. •T306A supports the involvement of a nucleophilic peroxo-anion during lyase activity. •The presence of cytochrome b{sub 5} augments C–C lyase activity. •Δ5-Steroids are preferred substrates for CYP17 catalysis. -- Abstract: Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon–carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17’s physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing

  4. Comparative research on activation technique for GaAs photocathodes

    NASA Astrophysics Data System (ADS)

    Chen, Liang; Qian, Yunsheng; Chang, Benkang; Chen, Xinlong; Yang, Rui

    2012-03-01

    The properties of GaAs photocathodes mainly depend on the material design and activation technique. In early researches, high-low temperature two-step activation has been proved to get more quantum efficiency than high-temperature single-step activation. But the variations of surface barriers for two activation techniques have not been well studied, thus the best activation temperature, best Cs-O ratio and best activation time for two-step activation technique have not been well found. Because the surface photovoltage spectroscopy (SPS) before activation is only in connection with the body parameters for GaAs photocathode such as electron diffusion length and the spectral response current (SRC) after activation is in connection with not only body parameters but also surface barriers, thus the surface escape probability (SEP) can be well fitted through the comparative research between SPS before activation and SEP after activation. Through deduction for the tunneling process of surface barriers by Schrödinger equation, the width and height for surface barrier I and II can be well fitted through the curves of SEP. The fitting results were well proved and analyzed by quantitative analysis of angle-dependent X-ray photoelectron spectroscopy (ADXPS) which can also study the surface chemical compositions, atomic concentration percentage and layer thickness for GaAs photocathodes. This comparative research method for fitting parameters of surface barriers through SPS before activation and SRC after activation shows a better real-time in system method for the researches of activation techniques.

  5. 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

  6. Characterization of the active site of ADP-ribosyl cyclase.

    PubMed

    Munshi, C; Thiel, D J; Mathews, I I; Aarhus, R; Walseth, T F; Lee, H C

    1999-10-22

    ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and

  7. New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase

    PubMed Central

    Cecchini, Davide A; Serra, Immacolata; Ubiali, Daniela; Terreni, Marco; Albertini, Alessandra M

    2007-01-01

    Background Immobilized Penicillin G Acylase (PGA) derivatives are biocatalysts that are industrially used for the hydrolysis of Penicillin G by fermentation and for the kinetically controlled synthesis of semi-synthetic β-lactam antibiotics. One of the most used supports for immobilization is glyoxyl-activated agarose, which binds the protein by reacting through its superficial Lys residues. Since in E. coli PGA Lys are also present near the active site, an immobilization that occurs through these residues may negatively affect the performance of the biocatalyst due to the difficult diffusion of the substrate into the active site. A preferential orientation of the enzyme with the active site far from the support surface would be desirable to avoid this problem. Results Here we report how it is possible to induce a preferential orientation of the protein during the binding process on aldehyde activated supports. A superficial region of PGA, which is located on the opposite side of the active site, is enriched in its Lys content. The binding of the enzyme onto the support is consequently forced through the Lys rich region, thus leaving the active site fully accessible to the substrate. Different mutants with an increasing number of Lys have been designed and, when active, immobilized onto glyoxyl agarose. The synthetic performances of these new catalysts were compared with those of the immobilized wild-type (wt) PGA. Our results show that, while the synthetic performance of the wt PGA sensitively decreases after immobilization, the Lys enriched mutants have similar performances to the free enzyme even after immobilization. We also report the observations made with other mutants which were unable to undergo a successful maturation process for the production of active enzymes or which resulted toxic for the host cell. Conclusion The desired orientation of immobilized PGA with the active site freely accessible can be obtained by increasing the density of Lys residues

  8. The aggregate site frequency spectrum (aSFS) for comparative population genomic inference

    PubMed Central

    Xue, Alexander T.; Hickerson, Michael J.

    2015-01-01

    Understanding how assemblages of species responded to past climate change is a central goal of comparative phylogeography and comparative population genomics, an endeavor that has increasing potential to integrate with community ecology. New sequencing technology now provides the potential to perform complex demographic inference at unprecedented resolution across assemblages of non-model species. To this end, we introduce the aggregate site frequency spectrum (aSFS), an expansion of the site frequency spectrum to use single nucleotide polymorphism (SNP) datasets collected from multiple, co-distributed species for assemblage-level demographic inference. We describe how the aSFS is constructed over an arbitrary number of independent population samples and then demonstrate how the aSFS can differentiate various multi-species demographic histories under a wide range of sampling configurations while allowing effective population sizes and expansion magnitudes to vary independently. We subsequently couple the aSFS with a hierarchical approximate Bayesian computation (hABC) framework to estimate degree of temporal synchronicity in expansion times across taxa, including an empirical demonstration with a dataset consisting of five populations of the threespine stickleback (Gasterosteus aculeatus). Corroborating what is generally understood about the recent post-glacial origins of these populations, the joint aSFS/hABC analysis strongly suggests that the stickleback data are most consistent with synchronous expansion after the Last Glacial Maximum (posterior probability = 0.99). The aSFS will have general application for multi-level statistical frameworks to test models involving assemblages and/or communities and as large-scale SNP data from non-model species become routine, the aSFS expands the potential for powerful next-generation comparative population genomic inference. PMID:26769405

  9. 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.

  10. Side by Side: What a Comparative Usability Study Told Us about a Web Site Redesign

    ERIC Educational Resources Information Center

    Dougan, Kirstin; Fulton, Camilla

    2009-01-01

    Library Web sites must compete against easy-to-use sites, such as Google Scholar, Google Books, and Wikipedia, for students' time and attention. Library Web sites must therefore be designed with aesthetics and user perceptions at the forefront. The Music and Performing Arts Library at Urbana-Champaign's Web site was overcrowded and in much need of…

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

    PubMed Central

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

    2015-01-01

    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 (Mw 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. Comparative neuroprotective properties of stilbene and catechin analogs: action via a plasma membrane receptor site?

    PubMed

    Bastianetto, Stéphane; Dumont, Yvan; Han, Yingshan; Quirion, Rémi

    2009-01-01

    Various studies have reported on the neuroprotective effects of polyphenols, widely present in food, beverages, and natural products. For example, we have shown that resveratrol, a polyphenol enriched in red wine and other foods such as peanuts, protects hippocampal cells against beta-amyloid (Abeta)-induced toxicity, a key protein involved in the neuropathology of Alzheimer disease. This effect involves, at least in part, the capacity of resveratrol to activate the phosphorylation of delta isoform of protein kinase C (PKC-delta). The neuroprotective action of resveratrol is shared by piceatannol, a stilbene derivative, as well as by tea-derived catechin gallate esters. The thioflavin T assay indicated that all these polyphenols inhibited the formation of Abeta fibrils, suggesting that this action likely also contributes to their neuroprotective effects. Binding and autoradiographic studies revealed that the effects of polyphenols might involve specific binding sites that are particularly enriched in the choroid plexus in the rat brain. Interestingly, the choroid plexus secretes transthyretin, a protein that has been shown to modulate Abeta aggregation and that may be critical to the maintenance of normal learning capacities in aging. Taken together, these data suggest that polyphenols target multiple enzymes/proteins, leading to their neuroprotective actions, possibly through action via specific plasma membrane binding sites.

  13. Comparing responses to horticultural-based and traditional activities in dementia care programs.

    PubMed

    Jarrott, Shannon E; Gigliotti, Christina M

    2010-12-01

    Engaging persons with dementia in meaningful activities supports well-being; however, care staff are challenged to implement age- and ability-appropriate activities in a group setting. We compared a randomly assigned treatment group, who received horticultural therapy-based (HT-based) programming to a comparison group, who engaged in traditional activities (TA) programming, on engagement and affect. Horticultural therapy-based programming was implemented twice weekly at 4 treatment sites for 6 weeks, while regular TA were observed at comparison sites. Results revealed no differences between groups on affective domains. Levels of adaptive behavior differed between the groups, with the treatment group demonstrating higher levels of active, passive, and other engagement and the comparison group demonstrating higher levels of self-engagement. Our results highlight the value of HT-based programs and the importance of simultaneously capturing participants' affective and behavioral responses. Theoretical and practical considerations about the facilitation of and context in which the programming occurs are discussed.

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

    PubMed Central

    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. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

  15. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    PubMed

    Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

    2015-09-25

    Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent.

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

    PubMed Central

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

    2010-01-01

    Decapping by Dcp2 is an essential step in 5′-3′ mRNA decay. In yeast, decapping requires an open-to-closed transition in Dcp2, though the link between closure and catalysis remains elusive. Here we show using NMR that cap binds conserved residues on both the catalytic and regulatory domains of Dcp2. Lesions in the cap-binding site on the regulatory domain reduce the catalytic step two orders of magnitude and block formation of the closed state whereas Dcp1 enhances the catalytic step by a factor of ten and promotes closure. We conclude that closure occurs during the rate-limiting catalytic step of decapping, juxtaposing the cap-binding region of each domain to form a composite active site. This work suggests a model for regulation of decapping, where coactivators trigger decapping by stabilizing a labile composite active site. PMID:20711189

  17. Comparing Participation in Activities among Children with Disabilities

    ERIC Educational Resources Information Center

    Masse, Louise C.; Miller, Anton R.; Shen, Jane; Schiariti, Veronica; Roxborough, Lori

    2012-01-01

    Introduction: Compared to typically developing peers, children with disabilities due to neurodevelopmental disorders and disabilities (NDD/D) and to chronic medical conditions (CMC) have reduced participation in activities. The extent to which these two groups of children have different levels of participation is unknown and was examined in this…

  18. The Ribotoxin Restrictocin Recognizes Its RNA Substrate by Selective Engagement of Active Site Residues

    PubMed Central

    2011-01-01

    Restrictocin and related fungal endoribonucleases from the α-sarcin family site-specifically cleave the sarcin/ricin loop (SRL) on the ribosome to inhibit translation and ultimately trigger cell death. Previous studies showed that the SRL folds into a bulged-G motif and tetraloop, with restrictocin achieving a specificity of ∼1000-fold by recognizing both motifs only after the initial binding step. Here, we identify contacts within the protein−RNA interface and determine the extent to which each one contributes to enzyme specificity by examining the effect of protein mutations on the cleavage of the SRL substrate compared to a variety of other RNA substrates. As with other biomolecular interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximum catalytic activity occurs when both motifs of the SRL are present, with the major contribution involving the bulged-G motif recognized by three lysine residues located adjacent to the active site: K110, K111, and K113. Our findings support a kinetic proofreading mechanism in which the active site residues H49 and, to a lesser extent, Y47 make greater catalytic contributions to SRL cleavage than to suboptimal substrates. This systematic and quantitative analysis begins to elucidate the principles governing RNA recognition by a site-specific endonuclease and may thus serve as a mechanistic model for investigating other RNA modifying enzymes. PMID:21417210

  19. Comparative anthelmintic activity investigation of selected ethno-medicinal weeds

    NASA Astrophysics Data System (ADS)

    Pueblos, Kirstin Rhys S.; Bajalla, Mark; Pacheco, Dixie; Ganot, Sheila; Paig, Daisy; Tapales, Radyn; Lagare, Jeanne; Quimque, Mark Tristan J.

    2017-01-01

    Helminth infections are one of the seriously neglected potent diseases in many parts of the world. The problems of parasitic helminthes becoming resistant to currently available anthelmintic drugs pose a challenge for the search - relying on natural products - for new and better anthelmintics. In this paper, four abundant Philippine weeds: Chrysopogon aciculatus Trin. Cyperus brevifolius Rottb., Ruellia tuberosa Linn. and Saccharum spontaneum Linn. were investigated for their anthelmintic activities to establish basis of their folkloric claim. The hexane-soluble and chloroform-soluble extracts were obtained through sequential solvent partitioning of the crude ethanolic extract of the air-dried aerial part of each plant sample. Meanwhile, the decoction was obtained from fresh aerial part of the plant samples. All extracts were then subjected to in vitro anthelmintic screening at different concentration as per method of Ghosh, et al. against African nightcrawler earthworms (Eudrillus euginiae) in which the activity of the extracts was determined by correlation with time. The anthelmintic bioassay results revealed a dose-dependent toxicity relationship. It indicated relatively low anthelmintic activities of the decoction of the four plant samples as compared to their corresponding crude ethanol extracts. Among the crude ethanol extracts, C. brevifolius (CBE) gave fastest time to bring about paralysis and death to the test organisms at all concentrations tested. For the hexane extracts, R. tuberosa (RTH) gave better activity among other plant samples. Lastly, among the chloroform-soluble extracts, both that of C. brevifolius (CBC) and R. tuberosa (RTC) comparably showed strongest anthelmintic activities at all tested concentrations, thus, exhibited best anthelmintic activity that is remarkably comparable to the positive control, Mebendazole at the highest concentration tested. In fact, CBC and RTC showed highest anthelmintic potential compared to all extracts tested in

  20. Seven-site versus three-site method of body composition using BodyMetrix ultrasound compared to dual-energy X-ray absorptiometry.

    PubMed

    Baranauskas, Marissa N; Johnson, Kelly E; Juvancic-Heltzel, Judith A; Kappler, Rachele M; Richardson, Laura; Jamieson, Scott; Otterstetter, Ronald

    2015-10-22

    Obesity is a steadily growing epidemic affecting all segments of the population including college-aged students. The weight gain that is evidenced amid the transitional stage of college years increases the risks associated with cardiovascular and metabolic diseases. The BodyMetrix® BX-2000 (ULTRA) using a seven-site method has been evaluated against dual-energy X-ray absorptiometry (DXA) for estimation of body composition, which has yielded conflicting results. To date, no studies have compared the three-site method Jackson and Pollock three-site method to DXA.

  1. Pathways from education to fertility decline: a multi-site comparative study.

    PubMed

    Snopkowski, Kristin; Towner, Mary C; Shenk, Mary K; Colleran, Heidi

    2016-04-19

    Women's education has emerged as a central predictor of fertility decline, but the many ways that education affects fertility have not been subject to detailed comparative investigation. Taking an evolutionary biosocial approach, we use structural equation modelling to examine potential pathways between education and fertility including: infant/child mortality, women's participation in the labour market, husband's education, social network influences, and contraceptive use or knowledge across three very different contexts: Matlab, Bangladesh; San Borja, Bolivia; and rural Poland. Using a comparable set of variables, we show that the pathways by which education affects fertility differ in important ways, yet also show key similarities. For example, we find that across all three contexts, education is associated with delayed age at first birth via increasing women's labour-force participation, but this pathway only influences fertility in rural Poland. In Matlab and San Borja, education is associated with lower local childhood mortality, which influences fertility, but this pathway is not important in rural Poland. Similarities across sites suggest that there are common elements in how education drives demographic transitions cross-culturally, but the differences suggest that local socioecologies also play an important role in the relationship between education and fertility decline.

  2. Solar irradiance computations compared with observations at the Baseline Surface Radiation Network Payerne site

    SciTech Connect

    Nowak, Daniela; Vuilleumier, Laurent; Long, Charles N.; Ohmura, Atsumu

    2008-07-18

    Radiative transfer model calculations of solar fluxes during cloud free periods often show considerable discrepancies with surface radiation observations. Many efforts have been undertaken to explain the differences between modeled and observed shortwave downward radiation (SDR). In this study, MODTRAN4v3r1TM (designed later simply as MODTRANTM) was used for model simulations and compared with high quality radiation observations of the Baseline Surface Radiation Network (BSRN) site at Payerne, Switzerland. Results are presented for cloud free shortwave downward radiation calculations. The median differences of modeled minus observed global SDR are small (< 1%) and within the instrumental error. The differences of modeled and observed direct and diffuse SDR show larger discrepancies of -1.8% and 5.2% respectively. The diffuse SDR is generally overestimated by the model and more important, the model to observation linear regression slope and zero-intercept differs significantly from their ideal values of 1 and 0. Possible reasons for the discrepancies are presented and discussed and some modifications are investigated for decreasing such differences between modeled and observed diffuse SDR. However, we could not resolve all the discrepancies. The best agreement is obtained when comparing model simulations whose 550nm aerosol optical depth input is inferred from observations using nine spectral channels, and using BSRN observations performed with a new and more precise shading disk and sun tracker system. In this case, the median bias between model simulations and observed diffuse SDR is -0.4 Wm-2 (< 1%).

  3. Interaction of aspartic acid-104 and proline-287 with the active site of m-calpain.

    PubMed Central

    Arthur, J S; Elce, J S

    1996-01-01

    In an ongoing study of the mechanisms of calpain catalysis and Ca(2+)-induced activation, the effects of Asp-104-->Ser and Pro-287-->Ser large subunit mutations on m-calpain activity, the pH-activity profile, Ca(2+)-sensitivity, and autolysis were measured. The importance of these positions was suggested by sequence comparisons between the calpain and papain families of cysteine proteinases. Asp-104 is adjacent to the active-site Cys-105, and Pro-287 is adjacent to the active-site Asn-286 and probably to the active-site His-262; both Asp-104 and Pro-287 are absolutely conserved in the known calpains, but are replaced by highly conserved serine residues in the papains. The single mutants had approx. 10-15% of wild-type activity, due mainly to a decrease in kcat, since Km was only slightly increased. The Pro-287-->Ser mutation appeared to cause a local perturbation of the catalytic Cys-105/His-262 catalytic ion pair, reducing its efficiency without major effect on the conformation and stability of the enzyme. The Asp-104-->Ser mutation caused a marked narrowing of the pH-activity curve, a 9-fold increase in Ca2+ requirement, and an acceleration of autolysis, when compared with the wild-type enzyme. The results indicated that Asp-104 alters the nature of its interaction with the catalytic ion pair during Ca(2+)-induced conformational change in calpain. This interaction may be direct or indirect, but is important in activation of the enzyme. PMID:8912692

  4. Characterization of Active Site Residues of Nitroalkane Oxidase†

    PubMed Central

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

    2010-01-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitrolkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Serl71 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by ~5-fold and decreases in the rate constant for product release of ~2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

  5. 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.

  6. 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.

  7. Comparative insecticide susceptibility and detoxification enzyme activities among pestiferous blattodea.

    PubMed

    Valles, S M; Koehler, P G; Brenner, R J

    1999-11-01

    Topical bioassays using propoxur, chlorpyrifos, and lambda-cyhalothrin were conducted on eight cockroach species. Based on lethal dose values, the relative toxicities of the insecticide classes were generally pyrethroid > carbamate > organophosphorous. Lambda-Cyhalothrin and propoxur were more toxic toward the Blattidae as compared with the Blattellidae. The order of lambda-cyhalothrin toxicity was Periplaneta americana > Periplaneta brunnea = Periplaneta australasiae = Periplaneta fuliginosa = Blatta orientalis > Blattella asahinai = Blattella germanica > Blattella vaga. The order of propoxur toxicity was B. orientalis > P. americana > P. brunnea = P. australasiae > B. asahinai > P. fuliginosa = B. germanica > B. vaga. The order of chlorpyrifos toxicity was P. americana > B. asahinai = B. vaga > B. orientalis = P. australasiae = P. brunnea > B. germanica = P. fuliginosa. Detoxification enzyme activities for each species also were measured and compared with insecticide toxicity. Propoxur LD50 was significantly (P = 0.01; r = 0.81) correlated with glutathione S-transferase activity. Lambda-Cyhalothrin LD50 correlated with methoxyresorufin O-demethylase activity (P = 0.01; r = 0.81), carboxylesterase activity (P = 0.03; r = - 0.75), general esterase activity (P = 0.02; r = - 0.79), and cockroach weight (P = 0.01; r = -0.95).

  8. 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.

  9. Structural role of the active-site metal in the conformation of Trypanosoma brucei phosphoglycerate mutase.

    PubMed

    Mercaldi, Gustavo F; Pereira, Humberto M; Cordeiro, Artur T; Michels, Paul A M; Thiemann, Otavio H

    2012-06-01

    Phosphoglycerate mutases (PGAMs) participate in both the glycolytic and the gluconeogenic pathways in reversible isomerization of 3-phosphoglycerate and 2-phosphoglycerate. PGAMs are members of two distinct protein families: enzymes that are dependent on or independent of the 2,3-bisphosphoglycerate cofactor. We determined the X-ray structure of the monomeric Trypanosoma brucei independent PGAM (TbiPGAM) in its apoenzyme form, and confirmed this observation by small angle X-ray scattering data. Comparing the TbiPGAM structure with the Leishmania mexicana independent PGAM structure, previously reported with a phosphoglycerate molecule bound to the active site, revealed the domain movement resulting from active site occupation. The structure reported here shows the interaction between Asp319 and the metal bound to the active site, and its contribution to the domain movement. Substitution of the metal-binding residue Asp319 by Ala resulted in complete loss of independent PGAM activity, and showed for the first time its involvement in the enzyme's function. As TbiPGAM is an attractive molecular target for drug development, the apoenzyme conformation described here provides opportunities for its use in structure-based drug design approaches. Database Structural data for the Trypanosoma brucei 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) has been deposited with the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank under code 3NVL.

  10. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 9 2012-07-01 2012-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing...

  11. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 9 2014-07-01 2014-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing...

  12. 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...

  13. 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...

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

    Code of Federal Regulations, 2013 CFR

    2013-01-01

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

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

    Code of Federal Regulations, 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...

  16. 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...

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

    SciTech Connect

    Teese, G.D.

    1995-09-28

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

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

    SciTech Connect

    Oliver, S. A.; Harris, V. G.; Hamdeh, H. H.; Ho, J. C.

    2000-05-08

    The cation site occupancy of a mechanically activated nanocrystalline zinc ferrite powder was determined as (Zn{sub 0.55}{sup 2+}Fe{sub 0.18}{sup 3+}){sub tet}[Zr{sub 0.45}{sup 2+}Fe{sub 1.82}{sup 3+}]{sub oct}O{sub 4} through analysis of extended x-ray absorption fine structure measurements, showing a large redistribution of cations between sites compared to normal zinc ferrite samples. The overpopulation of cations in the octahedral sites was attributed to the ascendance in importance of the ionic radii over the crystal energy and bonding coordination in determining which interstitial sites are occupied in this structurally disordered powder. Slight changes are observed in the local atomic environment about the zinc cations, but not the iron cations, with respect to the spinel structure. The presence of Fe{sup 3+} on both sites is consistent with the measured room temperature magnetic properties. (c) 2000 American Institute of Physics.

  19. 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.

  20. PARP promoter-mediated activation of a VSG expression site promoter in insect form Trypanosoma brucei.

    PubMed

    Urményi, T P; Van der Ploeg, L H

    1995-03-25

    In trypanosomes the rRNA, PARP and VSG gene promoters mediate alpha-amanitin-resistant transcription of protein coding genes, presumably by RNA polymerase (pol) I. We compared the activity of PARP and VSG promoters integrated at one of the alleles of the largest subunit of pol II genes in insect form trypanosomes. Even though both promoters are roughly equally active in transient transformation assays in insect form trypanosomes, only the PARP promoter functioned effectively when integrated at the pol II largest subunit or other loci. Promoter activity in transient transformation assays is therefore not necessarily predictive of transcriptional activity once integrated into the trypanosome genome. The integrated fully active PARP promoter could upregulate in cis an otherwise poorly active integrated VSG promoter. The PARP promoter nucleotide sequence elements responsible for VSG promoter activation coincided with most of the important PARP promoter elements mapped previously by linker scanning mutagenesis, indicating that it is not a single unique promoter element that was responsible for VSG promoter activation. The data suggest that PARP promoter-mediated activation of the VSG promoter does not result from complementation of the VSG promoter with a single insect form-specific transcription factor whose binding site is missing from the VSG promoter and present in the PARP promoter. We favor a model in which chromatin structure at the locus is altered by the PARP promoter, allowing VSG promoter activation in insect form trypanosomes. We discuss the significance of these observations for the control of VSG promoters in insect form trypanosomes.

  1. Lidar research activities and observations at NARL site, Gadanki, India

    NASA Astrophysics Data System (ADS)

    Yellapragada, Bhavani Kumar

    2016-05-01

    The National Atmospheric Research Laboratory (NARL), a unit of Department of Space (DOS), located at Gadanki village (13.5°N, 79.2°E, 370 m AMSL) in India, is involved in the development of lidar remote sensing technologies for atmospheric research. Several advanced lidar technologies employing micropulse, polarization, Raman and scanning have been developed at this site and demonstrated for atmospheric studies during the period between 2008 and 2015. The technology of micropulse lidar, operates at 532 nm wavelength, was successfully transferred to an industry and the commercial version has been identified for Indian Lidar network (I-LINK) programme. Under this lidar network activity, several lidar units were installed at different locations in India to study tropospheric aerosols and clouds. The polarization sensitive lidar technology was realized using a set of mini photomultiplier tube (PMT) units and has the capability to operate during day and night without a pause. The lidar technology uses a compact flashlamp pumped Qswitched laser and employs biaxial configuration between the transmitter and receiver units. The lidar technology has been utilized for understanding the polarization characteristics of boundary layer aerosols during the mixed layer development. The demonstrated Raman lidar technology, uses the third harmonic wavelength of Nd:YAG laser, provides the altitude profiles of aerosol backscattering, extinction and water vapor covering the boundary layer range and allows operation during nocturnal periods. The Raman lidar derived height profiles of aerosol backscattering and extinction coefficient, lidar ratio, and watervapor mixing ratio inform the tropical boundary layer aerosol characteristics. The scanning lidar technology uses a near infrared laser wavelength for probing the lower atmosphere and has been utilized for high resolution cloud profiling during convective periods. The lidar technology is also used for rain rate measurement during

  2. 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.

  3. Comparative kinetics of Qi site inhibitors of cytochrome bc1 complex: picomolar antimycin and micromolar cyazofamid.

    PubMed

    Li, Hui; Zhu, Xiao-Lei; Yang, Wen-Chao; Yang, Guang-Fu

    2014-01-01

    Antimycin and cyazofamid are specific inhibitors of the mitochondrial respiratory chain and bind to the Qi site of the cytochrome bc1 complex. With the aim to understand the detailed molecular inhibition mechanism of Qi inhibitors, we performed a comparative investigation of the inhibitory kinetics of them against the porcine bc1 complex. The results showed that antimycin is a slow tight-binding inhibitor of succinate-cytochrome c reductase (SCR) with Ki  = 0.033 ± 0.00027 nm and non-competitive inhibition with respect to cytochrome c. Cyazofamid is a classical inhibitor of SCR with Ki  = 12.90 ± 0.91 μm and a non-competitive inhibitor with respect to cytochrome c. Both of them show competitive inhibition with respect to substrate DBH2 . Further molecular docking and quantum mechanics calculations were performed. The results showed that antimycin underwent significant conformational change upon the binding. The energy barrier between the conformations in the crystal and in the binding pocket is ~13.63 kcal/mol. Antimycin formed an H-bond with Asp228 and two water-bridged H-bonds with Lys227 and His201, whereas cyazofamid formed only one H-bond with Asp228. The conformational change and the different hydrogen bonding network might account for why antimycin is a slow tight-binding inhibitor, whereas cyazofamid is a classic inhibitor.

  4. Microbicidal activity of monochloramine and chloramine T compared.

    PubMed

    Arnitz, R; Nagl, M; Gottardi, W

    2009-10-01

    Chloramine T (CAT) and monochloramine (NH2Cl) are active chlorine compounds and well-known biocides. CAT has stronger oxidative activity than NH(2)Cl, which is a smaller, more lipophilic molecule. The question arises whether lower oxidative activity can be compensated by higher lipophilicity. To address this problem, we investigated the bactericidal and fungicidal activity of pure NH(2)Cl compared to CAT. Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus fumigatus, A. flavus, and Candida albicans were subjected to quantitative killing assays at 20 degrees C and pH 7.1 in equimolar solutions of CAT or NH2Cl. NH2Cl was superior to CAT against all test strains at all test concentrations. At a concentration of 0.036 mM, NH2Cl reduced the count of E. coli (S. aureus) by 3log10 within 1 min (5 min), whereas CAT needed 120 min (30 min) for the same effect. At 0.107 mM NH2Cl, a 3log10 reduction of P. aeruginosa was achieved after 5 min compared to 20 min using CAT. NH2Cl (0.355 mM) caused a 2log10 reduction of C. albicans within 30 s, whereas 60 min were necessary for the same reduction with 0.355 mM CAT. The difference between the antiseptics was even more pronounced when tested on aspergilli. NH2Cl had a significantly stronger bactericidal and fungicidal activity than CAT despite its lower oxidative activity. This phenomenon can be attributed to its lipophilicity and smaller bulk, and it should be taken into account when developing and using chloramine antiseptics.

  5. Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP).

    PubMed

    Zhao, Hong-Yan; Wu, Li-Ying; Liu, Gang; Feng, Hong

    2016-12-01

    To engineer dehairing alkaline protease (DHAP) variants to improve cold activity and increase thermostability so these variants are suitable for the leather processing industry. Based on previous studies with bacterial alkaline proteases, double-site mutations (W106K/V149I and W106K/M124L) were introduced into the DHAP from Bacillus pumilus. Compared with the wild-type DHAP hydrolytic activity, the double-site variant W106K/V149I showed an increase in specific hydrolytic activity at 15 °C by 2.3-fold toward casein in terms of hydrolytic rate and 2.7-fold toward the synthetic peptide AAPF-pN by means of kcat/Km value. The thermostability of the variant (W106K/V149I) was improved with the half-life at 60 and 70 °C increased by 2.7- and 5.0-fold, respectively, when compared with the thermostability of the wild-type DHAP. Conclusively, an increase in the cold activity and thermostability of a bacterial alkaline protease was achieved by protein engineering.

  6. Comparative fitness assessment of Anopheles stephensi transgenic lines receptive to site-specific integration.

    PubMed

    Amenya, D A; Bonizzoni, M; Isaacs, A T; Jasinskiene, N; Chen, H; Marinotti, O; Yan, G; James, A A

    2010-04-01

    Genetically modified mosquitoes that are unable to transmit pathogens offer opportunities for controlling vector-borne diseases such as malaria and dengue. Site-specific gene recombination technologies are advantageous in the development of these insects because antipathogen effector genes can be inserted at integration sites in the genome that cause the least alteration in mosquito fitness. Here we describe Anopheles stephensi transgenic lines containing phi C31 attP'docking' sites linked to a fluorescent marker gene. Chromosomal insertion sites were determined and life-table parameters were assessed for transgenic mosquitoes of each line. No significant differences in fitness between the transgenic and nontransgenic mosquitoes were detected in this study. These transgenic lines are suitable for future site-specific integrations of antiparasite transgenes into the attP sites.

  7. Pose prediction accuracy in docking studies and enrichment of actives in the active site of GSK-3beta.

    PubMed

    Gadakar, Pravin Kumar; Phukan, Samiron; Dattatreya, Prasanna; Balaji, V N

    2007-01-01

    We present molecular docking studies on the inhibitors of GSK-3beta kinase in the enzyme binding sites of the X-ray complexes (1H8F, 1PYX, 1O9U, 1Q4L, 1Q5K, and 1UV5) using the Schrödinger docking tool Glide. Cognate and cross-docking studies using standard precision (SP) and extraprecision (XP) algorithms have been carried out. Cognate docking studies demonstrate that docked poses similar to X-ray poses (root-mean-square deviations of less than 2 A) are found within the top four ranks of the GlideScore and E-model scores. However, cross-docking studies typically produce poses that are significantly deviated from X-ray poses in all but a couple of cases, implying potential for induced fit effects in ligand binding. In this light, we have also carried out induced fit docking studies in the active sites of 1O9U, 1Q4L, and 1Q5K. Specifically, conformational changes have been effected in the active sites of these three protein structures to dock noncognate ligands. Thus, for example, the active site of 1O9U has been induced to fit the ligands of 1Q4L, 1Q5K, and 1UV5. These studies produce ligand docked poses which have significantly lower root-mean-square deviations relative to their X-ray crystallographic poses, when compared to the corresponding values from the cross-docking studies. Furthermore, we have used an ensemble of the induced fit models and X-ray structures to enhance the retrieval of active GSK-3beta inhibitors seeded in a decoy database, normally used in Glide validation studies. Thus, our studies provide valuable insights into computational strategies useful for the identification of potential GSK-3beta inhibitors.

  8. Active-site zinc ligands and activated H2O of zinc enzymes.

    PubMed Central

    Vallee, B L; Auld, D S

    1990-01-01

    The x-ray crystallographic structures of 12 zinc enzymes have been chosen as standards of reference to identify the ligands to the catalytic and structural zinc atoms of other members of their respective enzyme families. Universally, H2O is a ligand and critical component of the catalytically active zinc sites. In addition, three protein side chains bind to the catalytic zinc atom, whereas four protein ligands bind to the structural zinc atom. The geometry and coordination number of zinc can vary greatly to accommodate particular ligands. Zinc forms complexes with nitrogen and oxygen just as readily as with sulfur, and this is reflected in catalytic zinc sites having a binding frequency of His much greater than Glu greater than Asp = Cys, three of which bind to the metal atom. The systematic spacing between the ligands is striking. For all catalytic zinc sites except the coenzyme-dependent alcohol dehydrogenase, the first two ligands are separated by a "short-spacer" consisting of 1 to 3 amino acids. These ligands are separated from the third ligand by a "long spacer" of approximately 20 to approximately 120 amino acids. The spacer enables formation of a primary bidentate zinc complex, whereas the long spacer contributes flexibility to the coordination sphere, which can poise the zinc for catalysis as well as bring other catalytic and substrate binding groups into apposition with the active site. The H2O is activated by ionization, polarization, or poised for displacement. Collectively, the data imply that the preferred mechanistic pathway for activating the water--e.g., zinc hydroxide or Lewis acid catalysis--will be determined by the identity of the other three ligands and their spacing. Images PMID:2104979

  9. Increased Rates of Long-Term Complications after MammoSite Brachytherapy Compared with Whole Breast Radiation Therapy

    PubMed Central

    Rosenkranz, Kari M; Tsui, Edmund; McCabe, Elizabeth B; Gui, Jiang; Underhill, Kelly; Barth, Richard J

    2013-01-01

    BACKGROUND Due to its short duration of therapy and low rates of local recurrence, women undergoing breast conservation are increasingly opting for partial breast irradiation with the MammoSite (Cytyc/Hologic) catheter. In early follow-up studies, few complications were reported. Few data, however, exist regarding longer-term complications. We compared the long-term local toxicities of MammoSite partial breast irradiation with those resulting from whole breast radiation. STUDY DESIGN This was a retrospective study performed in a single academic medical center. All patients who underwent breast-conserving surgery between 2003 and 2008, who met institutional criteria for brachytherapy, were included. We compared women treated with MammoSite with patients treated with whole breast radiation therapy (WBRT). Endpoints included incidence of palpable masses at the lumpectomy site, telangiectasias, and local recurrence. RESULTS Seventy-one MammoSite patients and 245 WBRT patients were well matched with regard to clinical characteristics. Median follow-up was 4 years. A palpable mass developed at the site of lumpectomy in 27% of the MammoSite patients compared with 7% of the WBRT patients (p < 0.0001). Telangiectasias developed more frequently in the MammoSite group than in the WBRT group (24% vs 4%, p < 0.001). Forty-two percent of patients treated with MammoSite developed a palpable mass, telangectasia, or both. CONCLUSIONS Palpable masses and telangiectasias are frequent long-term complications after MammoSite brachytherapy and occur at a significantly higher rate after MammoSite brachytherapy than after WBRT. This increased rate of long-term local toxicity should be considered when counseling women on options for adjuvant radiation therapy after breast-conserving surgery. PMID:23830216

  10. Comparative pharmacological activity of optical isomers of phenibut.

    PubMed

    Dambrova, Maija; Zvejniece, Liga; Liepinsh, Edgars; Cirule, Helena; Zharkova, Olga; Veinberg, Grigory; Kalvinsh, Ivars

    2008-03-31

    Phenibut (3-phenyl-4-aminobutyric acid) is a GABA (gamma-aminobutyric acid)-mimetic psychotropic drug which is clinically used in its racemic form. The aim of the present study was to compare the effects of racemic phenibut and its optical isomers in pharmacological tests and GABAB receptor binding studies. In pharmacological tests of locomotor activity, antidepressant and pain effects, S-phenibut was inactive in doses up to 500 mg/kg. In contrast, R-phenibut turned out to be two times more potent than racemic phenibut in most of the tests. In the forced swimming test, at a dose of 100 mg/kg only R-phenibut significantly decreased immobility time. Both R-phenibut and racemic phenibut showed analgesic activity in the tail-flick test with R-phenibut being slightly more active. An GABAB receptor-selective antagonist (3-aminopropyl)(diethoxymethyl)phosphinic acid (CGP35348) inhibited the antidepressant and antinociceptive effects of R-phenibut, as well as locomotor depressing activity of R-phenibut in open field test in vivo. The radioligand binding experiments using a selective GABAB receptor antagonist [3H]CGP54626 revealed that affinity constants for racemic phenibut, R-phenibut and reference GABA-mimetic baclofen were 177+/-2, 92+/-3, 6.0+/-1 microM, respectively. We conclude that the pharmacological activity of racemic phenibut relies on R-phenibut and this correlates to the binding affinity of enantiomers of phenibut to the GABAB receptor.

  11. Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

    PubMed

    Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

    2015-12-11

    Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases.

  12. 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

  13. The yeast regulator of transcription protein Rtr1 lacks an active site and phosphatase activity.

    PubMed

    Xiang, Kehui; Manley, James L; Tong, Liang

    2012-07-10

    The activity of RNA polymerase II (Pol II) is controlled in part by the phosphorylation state of the C-terminal domain (CTD) of its largest subunit. Recent reports have suggested that yeast regulator of transcription protein, Rtr1, and its human homologue RPAP2, possess Pol II CTD Ser5 phosphatase activity. Here we report the crystal structure of Kluyveromyces lactis Rtr1, which reveals a new type of zinc finger protein and does not have any close structural homologues. Importantly, the structure does not show evidence of an active site, and extensive experiments to demonstrate its CTD phosphatase activity have been unsuccessful, suggesting that Rtr1 has a non-catalytic role in CTD dephosphorylation.

  14. rVISTA for Comparative Sequence-Based Discovery of Functional Transcription Factor Binding Sites

    SciTech Connect

    Loots, Gabriela G.; Ovcharenko, Ivan; Pachter, Lior; Dubchak, Inna; Rubin, Edward M.

    2002-03-08

    Identifying transcriptional regulatory elements represents a significant challenge in annotating the genomes of higher vertebrates. We have developed a computational tool, rVISTA, for high-throughput discovery of cis-regulatory elements that combines transcription factor binding site prediction and the analysis of inter-species sequence conservation. Here, we illustrate the ability of rVISTA to identify true transcription factor binding sites through the analysis of AP-1 and NFAT binding sites in the 1 Mb well-annotated cytokine gene cluster1 (Hs5q31; Mm11). The exploitation of orthologous human-mouse data set resulted in the elimination of 95 percent of the 38,000 binding sites predicted upon analysis of the human sequence alone, while it identified 87 percent of the experimentally verified binding sites in this region.

  15. Performance assessment of the Greater Confinement Disposal facility on the Nevada Test Site: Comparing the performance of two conceptual site models

    SciTech Connect

    Baer, T.A.; Price, L.L.; Gallegos, D.P.

    1993-12-31

    A small amount of transuranic (TRU) waste has been disposed of at the Greater Confinement Disposal (GCD) site located on the Nevada Test Site`s (NTS) Radioactive Waste Management Site (RWMS). The waste has been buried in several deep (37 m) boreholes dug into the floor of an alluvial basin. For the waste to remain in its current configuration, the DOE must demonstrate compliance of the site with the TRU disposal requirements, 40 CFR 191. Sandia`s approach to process modelling in performance assessment is to use demonstrably conservative models of the site. Choosing the most conservative model, however, can be uncertain. As an example, diffusion of contaminants upward from the buried waste in the vadose zone water is the primary mechanism of release. This process can be modelled as straight upward planar diffusion or as spherical diffusion in all directions. The former has high fluxes but low release areas, the latter has lower fluxes but is spread over a greater area. We have developed analytic solutions to a simple test problem for both models and compared the total integrated discharges. The spherical diffusion conceptual model results in at least five times greater release to the accessible environment than the planar model at all diffusivities. Modifying the planar model to allow for a larger release, however, compensated for the smaller original planar discharge and resulted in a new planar model that was more conservative that the spherical model except at low diffusivities.

  16. Comparative antimycobacterial activities of ofloxacin, ciprofloxacin and grepafloxacin.

    PubMed

    Vacher, S; Pellegrin, J L; Leblanc, F; Fourche, J; Maugein, J

    1999-11-01

    Infections caused by non-tuberculous mycobacteria and multidrug-resistant Mycobacterium tuberculosis are difficult to treat. New compounds potentially active against these bacteria are therefore constantly being sought. Among them is grepafloxacin, a new C5 fluoroquinolone. A panel of 130 isolates of mycobacteria including 33 M. tuberculosis isolates and 97 isolates of different species of atypical mycobacteria were analysed for susceptibility to grepafloxacin, ofloxacin and ciprofloxacin. The MICs of these fluoroquinolones were determined using the agar-dilution method. Different mycobacterial species showed different degrees of susceptibility to grepafloxacin, ofloxacin and ciprofloxacin but little difference was observed between the MICs of the three antibiotics against strains of the same mycobacterial species. In addition, to evaluate the intracellular activity of these drugs, six strains of mycobacteria were studied using a human-macrophage infection model. Preliminary results of macrophage experiments showed that grepafloxacin was more active than ofloxacin and ciprofloxacin, particularly against Mycobacterium kansasii and, to a lesser degree, against Mycobacterium avium complex and Mycobacterium marinum. However, the three fluoroquinolones had comparable activities against M. tuberculosis.

  17. Antianaerobic activity of sulopenem compared to six other agents.

    PubMed

    Ednie, Lois M; Appelbaum, Peter C

    2009-05-01

    Agar dilution MIC methodology was used to compare the activity of sulopenem with those of amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin-tazobactam, imipenem, clindamycin, and metronidazole against 431 anaerobes. Overall, MIC(50)/(90) values were as follows: sulopenem, 0.25/1.0 microg/ml; amoxicillin/clavulanate, 0.5/2.0 microg/ml; ampicillin/sulbactam, 0.5/4.0 microg/ml; piperacillin/tazobactam, 0.25/8.0 microg/ml; imipenem, 0.06/1.0 microg/ml; clindamycin, 0.25/16.0 microg/ml; and metronidazole, 1.0/4.0 microg/ml.

  18. Comparative studies of brain activation with MEG and functional MRI

    SciTech Connect

    George, J.S.; Aine, C.J.; Sanders, J.A.; Lewine, J.D.; Caprihan, A.

    1993-12-31

    The past two years have witnessed the emergence of MRI as a functional imaging methodology. Initial demonstrations involved the injection of a paramagnetic contrast agent and required ultrafast echo planar imaging capability to adequately resolve the passage of the injected bolus. By measuring the local reduction in image intensity due to magnetic susceptibility, it was possible to calculate blood volume, which changes as a function of neural activation. Later developments have exploited endogenous contrast mechanisms to monitor changes in blood volume or in venous blood oxygen content. Recently, we and others have demonstrated that it is possible to make such measurements in a clinical imager, suggesting that the large installed base of such machines might be utilized for functional imaging. Although it is likely that functional MRI (fMRI) will subsume some of the clinical and basic neuroscience applications now touted for MEG, it is also clear that these techniques offer different largely complementary, capabilities. At the very least, it is useful to compare and cross-validate the activation maps produced by these techniques. Such studies will be valuable as a check on results of neuromagnetic distributed current reconstructions and will allow better characterization of the relationship between neurophysiological activation and associated hemodynamic changes. A more exciting prospect is the development of analyses that combine information from the two modalities to produce a better description of underlying neural activity than is possible with either technique in isolation. In this paper we describe some results from initial comparative studies and outline several techniques that can be used to treat MEG and fMRI data within a unified computational framework.

  19. Discovery of Active Hydrothermal Sites Along the Mariana Volcanic Arc, Western Pacific Ocean

    NASA Astrophysics Data System (ADS)

    Baker, E. T.; Embley, R. W.; Resing, J. A.; Lupton, J. E.; Massoth, G. J.; de Ronde, C. E.; Nakamura, K.; Walker, S. L.

    2003-12-01

    Some 20,000 km of volcanic arcs, roughly one-third the total length of the global midocean ridge (MOR) system, rim the western Pacific Ocean. But compared to 25 years of hydrothermal investigations along MORs, exploration of similar activity on the estimated 600 submarine arc volcanoes is only beginning. In February 2003, as part of the Submarine Ring of Fire project funded by NOAA's Ocean Exploration Program, we made the first systematic survey of hydrothermal activity along the 1270-km-long Mariana intraoceanic volcanic arc, which lies almost entirely within the US EEZ. Prior fieldwork had documented active (but low-temperature) hydrothermal discharge on only three volcanoes: Kasuga 2, Kasuga 3, and Esmeralda Bank. During the cruise, we conducted 70 CTD operations over more than 50 individual volcanoes from 13° N to 23° N, plus a continuous CTD survey along 75 km of the back-arc spreading center (13° 15'N to 13° 41'N) adjacent to the southern end of the arc. We found evidence for active hydrothermal venting at 11 submarine volcanoes with summit (or caldera floor) depths ranging from 50 to 1550 m. Two additional sites were identified on the back-arc spreading center. Ongoing analyses of collected water samples could increase these totals. Our results confirmed continuing hydrothermal activity at Kasuga 2 (but not Kasuga 3) and Esmeralda Bank, in addition to newly discovered sites on nine other volcanoes. Many of these sites produce intense and widely dispersed plumes indicative of vigorous, high-temperature discharge. The volcanoes with active hydrothermal systems are about equally divided between those with and without summit calderas. The addition of the Marianas data greatly improves our view of hydrothermal sources along arcs. The 20,000 km of Pacific arcs can be divided between 6380 km of intraoceanic (i.e., mostly submarine) arcs and 13,880 km of island (i.e., mostly subaerial) arcs. At present, ˜15% of the total length of Pacific arcs has been surveyed

  20. Site-directed mutagenesis of the Anabaena sp. strain PCC 7120 nitrogenase active site to increase photobiological hydrogen production.

    PubMed

    Masukawa, Hajime; Inoue, Kazuhito; Sakurai, Hidehiro; Wolk, C Peter; Hausinger, Robert P

    2010-10-01

    Cyanobacteria use sunlight and water to produce hydrogen gas (H₂), which is potentially useful as a clean and renewable biofuel. Photobiological H₂ arises primarily as an inevitable by-product of N₂ fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H₂ production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N₂. Most of the 49 variants examined were deficient in N₂-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N₂ atmosphere significantly increased their in vivo rates of H₂ production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H₂ compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H₂ in an aerobic, nitrogen-containing environment.

  1. CO-dynamics in the active site of cytochrome c oxidase

    NASA Astrophysics Data System (ADS)

    Soloviov, Maksym; Meuwly, Markus

    2014-04-01

    The transfer of CO from heme a3 to the CuB site in Cytochrome c oxidase (CcO) after photolysis is studied using molecular dynamics simulations using an explicitly reactive, parametrized potential energy surface based on density functional theory calculations. After photodissociation from the heme-Fe, the CO ligand rebinds to the CuB site on the sub-picosecond time scale. Depending on the simulation protocol the characteristic time ranges from 260 fs to 380 fs which compares with an estimated 450 fs from experiment based on the analysis of the spectral changes as a function of time delay after the photodissociating pulse. Following photoexcitation ≈90% of the ligands are found to rebind to either the CuB (major component, 85%) or the heme-Fe (minor component, 2%) whereas about 10% remain in an unbound state. The infrared spectra of unbound CO in the active site is broad and featureless and no appreciable shift relative to gas-phase CO is found, which is in contrast to the situation in myoglobin. These observations explain why experimentally, unbound CO in the binuclear site of CcO has not been found as yet.

  2. Thermal regime of active layer at two lithologically contrasting sites on James Ross Island, Antarctic Peninsula.

    NASA Astrophysics Data System (ADS)

    Hrbáček, Filip; Nývlt, Daniel; Láska, Kamil

    2016-04-01

    Antarctic Peninsula region (AP) represents one of the most rapidly warming parts of our planet in the last 50 years. Despite increasing research activities along both western and eastern sides of AP in last decades, there is still a lot of gaps in our knowledge relating to permafrost, active layer and its thermal and physical properties. This study brings new results of active layer monitoring on James Ross Island, which is the largest island in northern AP. Its northern part, Ulu Peninsula, is the largest ice-free area (more than 200 km2) in the region. Due its large area, we focused this study on sites located in different lithologies, which would affect local thermal regime of active layer. Study site (1) at Abernethy Flats area (41 m a.s.l.) lies ~7 km from northern coast. Lithologically is formed by disintegrated Cretaceous calcareous sandstones and siltstones of the Santa Marta Formation. Study site (2) is located at the northern slopes of Berry Hill (56 m a.s.l.), about 0.4 km from northern coastline. Lithology is composed of muddy to intermediate diamictites, tuffaceous siltstones to fine grained sandstones of the Mendel Formation. Data of air temperature at 2 meters above ground and the active layer temperatures at 75 cm deep profiles were obtained from both sites in period 1 January 2012 to 31 December 2014. Small differences were found when comparing mean air temperatures and active temperatures at 5 and 75 cm depth in the period 2012-2014. While the mean air temperatures varied between -7.7 °C and -7.0 °C, the mean ground temperatures fluctuated between -6.6 °C and -6.1 °C at 5 cm and -6.9 °C and -6.0 °C at 75 cm at Abernethy Flats and Berry Hill slopes respectively. Even though ground temperature differences along the profiles weren't pronounced during thawing seasons, the maximum active layer thickness was significantly larger at Berry Hill slopes (80 to 82 cm) than at Abernethy Flats (52 to 64 cm). We assume this differences are affected by

  3. 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)

  4. Single-site Laparoscopic Colorectal Surgery Provides Similar Clinical Outcomes Compared to Standard Laparoscopic Surgery: An Analysis of 626 Patients

    PubMed Central

    Sangster, William; Messaris, Evangelos; Berg, Arthur S.; Stewart, David B.

    2015-01-01

    BACKGROUND Compared to standard laparoscopy, single-site laparoscopic colorectal surgerymay potentially offer advantages by creating fewer surgical incisions and providing a multi-functional trocar. Previous comparisons, however, have been limited by small sample sizes and selection bias. OBJECTIVE To compare 60-day outcomes between standard laparoscopic and single-site laparoscopic colorectal surgery patients undergoing elective and urgent surgeries. DESIGN This was an unselected retrospective cohort study comparing patients who underwent elective and unplanned standard laparoscopic or single-site laparoscopic colorectal resections for benign and malignant disease between 2008 and 2014. Outcomes were compared using univariate analyses. SETTING This study was conducted at a single institution. PATIENTS A total of 626 consecutive patients undergoing laparoscopic colorectal surgery were included. MAIN OUTCOME MEASURES Morbidity and mortality within 60 postoperative days. RESULTS 318 (51%) and 308 (49%) patients underwent standard laparoscopic and single-site laparoscopic procedures, respectively. No significant difference was noted in mean operative time (Standard laparoscopy 182.1 ± 81.3 vs. Single-site laparoscopy 177±86.5, p=0.30) and postoperative length of stay (Standard laparoscopy 4.8±3.4 vs. Single-site laparoscopy 5.5 ± 6.9, p=0.14). Conversions to laparotomy and 60-day readmissions were also similar for both cohorts across all procedures performed. A significant difference was identified in the number of patients who developed postoperative complications (Standard laparoscopy 19.2% vs. Single-site laparoscopy 10.7%, p=0.004), especially with respect to surgical-site infections (Standard laparoscopy 11.3% vs. Single-site laparoscopy 5.8%, p=0.02). LIMITATIONS This was a retrospective, single institution study. CONCLUSIONS Single-site laparoscopic colorectal surgery demonstrates similar results to standard laparoscopic colorectal surgery in regards to

  5. Comparative evaluation of leachate pollution index of MSW landfill site of Kolkata with other metropolitan cities of India.

    PubMed

    Motling, Sanjay; Dutta, Amit; Mukherjee, S N; Kumar, Sunil

    2013-07-01

    The uncontrolled tipping of mixed urban solid waste in landfill site causes serious negative impacts on the environment. The major issue in this context is the generation of leachate which possesses potential of polluting freshwater ecosystem including groundwater besides associated health hazards and depletion of soil fertility. In this context, a pseudo computation quantitative tool, known as leachate pollution index (LPI), has been developed by some researchers for scaling pollution potential of landfill site owing to emergence of leachate. This paper. deals with the assessment of leachate quality of existing landfill site of Kolkata situated at Dhapa waste dumping ground through evaluation of the LPI from experimental analysis of leachate. The leachate was collected from this site in different seasons. 18 parameters were tested with real leachate samples in the Environmental Engineering Laboratory of Civil Engineering Department of Jadavpur University Kolkata. The results exhibited a very high value of organic pollutants in the leachate with COD as 21,129 mg/L and also values of TDS, Fe2+, Cr, Zn, chloride and ammonical nitrogen. The LPI value of Kolkata landfill site at Dhapa was estimated and also compared with leachate quality data of other metropolitan cities viz. Mumbai, Delhi, Chennai as available in literatures. It is found that LPI of the Kolkata landfill site is highest compared to all other landfill sites of other metropolitan cities in India.

  6. 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.

  7. Revealing the nature of the active site on the carbon catalyst for C-H bond activation.

    PubMed

    Sun, XiaoYing; Li, Bo; Su, Dangsheng

    2014-09-28

    A reactivity descriptor for the C-H bond activation on the nanostructured carbon catalyst is proposed. Furthermore the calculations reveal that the single ketone group can be an active site in ODH reaction.

  8. 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.

  9. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2009-09-01

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

  11. 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.

  12. Structural characterization of single nucleotide variants at ligand binding sites and enzyme active sites of human proteins

    PubMed Central

    Yamada, Kazunori D.; Nishi, Hafumi; Nakata, Junichi; Kinoshita, Kengo

    2016-01-01

    Functional sites on proteins play an important role in various molecular interactions and reactions between proteins and other molecules. Thus, mutations in functional sites can severely affect the overall phenotype. Progress of genome sequencing projects has yielded a wealth of information on single nucleotide variants (SNVs), especially those with less than 1% minor allele frequency (rare variants). To understand the functional influence of genetic variants at a protein level, we investigated the relationship between SNVs and protein functional sites in terms of minor allele frequency and the structural position of variants. As a result, we observed that SNVs were less abundant at ligand binding sites, which is consistent with a previous study on SNVs and protein interaction sites. Additionally, we found that non-rare variants tended to be located slightly apart from enzyme active sites. Examination of non-rare variants revealed that most of the mutations resulted in moderate changes of the physico-chemical properties of amino acids, suggesting the existence of functional constraints. In conclusion, this study shows that the mapping of genetic variants on protein structures could be a powerful approach to evaluate the functional impact of rare genetic variations. PMID:27924270

  13. Seismic hazard for the Savannah River Site: A comparative evaluation of the EPRI and LLNL assessments

    SciTech Connect

    Wingo, H.E.

    1992-05-20

    This report was conducted to: (1) develop an understanding of causes for the vast differences between the two comprehensive studies, and (2) using a methodology consistent with the reconciled methods employed in the two studies, develop a single seismic hazard for the Savannah River Site suitable for use in seismic probabilistic risk assessments with emphasis on the K Reactor. Results are presented for a rock site which is a typical because detailed evaluations of soil characteristics at the K Reactor are still in progress that account for the effects of a soil stablizing grouting program. However when the soils analysis is completed, the effects of soils can be included with this analysis with the addition of a single factor that will decrease slightly the seismic hazard for a rock site.

  14. Lamellipodial actin mechanically links myosin activity with adhesion site formation

    PubMed Central

    Giannone, Gregory; Dubin-Thaler, Benjamin; Rossier, Olivier; Cai, Yunfei; Chaga, Oleg; Jiang, Guoying; Beaver, William; Döbereiner, Hans-Günther; Freund, Yoav; Borisy, Gary; Sheetz, Michael P.

    2013-01-01

    Summary Cell motility proceeds by cycles of edge protrusion, adhesion and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process. PMID:17289574

  15. Antianaerobic Activity of Sulopenem Compared to Six Other Agents ▿

    PubMed Central

    Ednie, Lois M.; Appelbaum, Peter C.

    2009-01-01

    Agar dilution MIC methodology was used to compare the activity of sulopenem with those of amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin-tazobactam, imipenem, clindamycin, and metronidazole against 431 anaerobes. Overall, MIC50/90 values were as follows: sulopenem, 0.25/1.0 μg/ml; amoxicillin/clavulanate, 0.5/2.0 μg/ml; ampicillin/sulbactam, 0.5/4.0 μg/ml; piperacillin/tazobactam, 0.25/8.0 μg/ml; imipenem, 0.06/1.0 μg/ml; clindamycin, 0.25/16.0 μg/ml; and metronidazole, 1.0/4.0 μg/ml. PMID:19223615

  16. 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.

  17. Characterization of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus: enzymatic activity and active site structure.

    PubMed

    Terasaka, Erina; Okada, Norihiro; Sato, Nozomi; Sako, Yoshihiko; Shiro, Yoshitsugu; Tosha, Takehiko

    2014-07-01

    Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating a more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

  18. Comparing measured and modelled soil carbon: which site-specific variables are linked to high stability?

    NASA Astrophysics Data System (ADS)

    Robertson, Andy; Schipanski, Meagan; Ma, Liwang; Ahuja, Lajpat; McNamara, Niall; Smith, Pete; Davies, Christian

    2016-04-01

    Changes in soil carbon (C) stocks have been studied in depth over the last two decades, as net greenhouse gas (GHG) sinks are highlighted to be a partial solution to the causes of climate change. However, the stability of this soil C is often overlooked when measuring these changes. Ultimately a net sequestration in soils is far less beneficial if labile C is replacing more stable forms. To date there is no accepted framework for measuring soil C stability, and as a result there is considerable uncertainty associated with the simulated impacts of land management and land use change when using process-based systems models. However, a recent effort to equate measurable soil C fractions to model pools has generated data that help to assess the impacts of land management, and can ultimately help to reduce the uncertainty of model predictions. Our research compiles this existing fractionation data along with site metadata to create a simplistic statistical model able to quantify the relative importance of different site-specific conditions. Data was mined from 23 published studies and combined with original data to generate a dataset of 100+ land use change sites across Europe. For sites to be included they required soil C fractions isolated using the Zimmermann et al. (2007) method and specific site metadata (mean annual precipitation, MAP; mean annual temperature, MAT; soil pH; land use; altitude). Of the sites, 75% were used to develop a generalized linear mixed model (GLMM) to create coefficients where site parameters can be used to predict influence on the measured soil fraction C stocks. The remaining 25% of sites were used to evaluate uncertainty and validate this empirical model. Further, four of the aforementioned sites were used to simulate soil C dynamics using the RothC, DayCent and RZWQM2 models. A sensitivity analysis (4096 model runs for each variable applying Latin hypercube random sampling techniques) was then used to observe whether these models place

  19. Comparative antipneumococcal activities of sulopenem and other drugs.

    PubMed

    Kosowska-Shick, Klaudia; Ednie, Lois M; McGhee, Pamela; Appelbaum, Peter C

    2009-06-01

    For 297 penicillin-susceptible, -intermediate, and -resistant pneumococcal strains, the sulopenem MIC(50)s were 0.008, 0.06, and 0.25, respectively, and the sulopenem MIC(90)s were 0.016, 0.25, and 0.5 microg/ml, respectively. The MIC(50)s of amoxicillin for the corresponding strains were 0.03, 0.25, and 2.0 microg/ml, respectively, and the MIC(90)s were 0.03, 1.0, and 8.0 microg/ml, respectively. The combination of amoxicillin and clavulanate gave MICs similar to those obtained with amoxicillin alone. The sulopenem MICs were similar to those of imipenem and meropenem. The MICs of ss-lactams increased with those of penicillin G, and among the quinolones tested, moxifloxacin had the lowest MICs. Additionally, 45 strains of drug-resistant type 19A pneumococci were tested by agar dilution and gave sulopenem MIC(50)s and MIC(90)s of 1.0 and 2.0 microg/ml, respectively. Both sulopenem and amoxicillin (with and without clavulanate) were bactericidal against all 12 strains tested at 2x MIC after 24 h. Thirty-one strains from 10 countries with various penicillin, amoxicillin, and carbapenems MICs, including those with the highest sulopenem MICs, were selected for sequencing analysis of the pbp1a, pbp2x, and pbp2b regions encoding the transpeptidase active site and MurM. We did not find any correlations between specific penicillin-binding protein-MurM patterns and changes in the MICs.

  20. Comparative Antipneumococcal Activities of Sulopenem and Other Drugs▿

    PubMed Central

    Kosowska-Shick, Klaudia; Ednie, Lois M.; McGhee, Pamela; Appelbaum, Peter C.

    2009-01-01

    For 297 penicillin-susceptible, -intermediate, and -resistant pneumococcal strains, the sulopenem MIC50s were 0.008, 0.06, and 0.25, respectively, and the sulopenem MIC90s were 0.016, 0.25, and 0.5 μg/ml, respectively. The MIC50s of amoxicillin for the corresponding strains were 0.03, 0.25, and 2.0 μg/ml, respectively, and the MIC90s were 0.03, 1.0, and 8.0 μg/ml, respectively. The combination of amoxicillin and clavulanate gave MICs similar to those obtained with amoxicillin alone. The sulopenem MICs were similar to those of imipenem and meropenem. The MICs of ß-lactams increased with those of penicillin G, and among the quinolones tested, moxifloxacin had the lowest MICs. Additionally, 45 strains of drug-resistant type 19A pneumococci were tested by agar dilution and gave sulopenem MIC50s and MIC90s of 1.0 and 2.0 μg/ml, respectively. Both sulopenem and amoxicillin (with and without clavulanate) were bactericidal against all 12 strains tested at 2× MIC after 24 h. Thirty-one strains from 10 countries with various penicillin, amoxicillin, and carbapenems MICs, including those with the highest sulopenem MICs, were selected for sequencing analysis of the pbp1a, pbp2x, and pbp2b regions encoding the transpeptidase active site and MurM. We did not find any correlations between specific penicillin-binding protein-MurM patterns and changes in the MICs. PMID:19307366

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

    PubMed

    Stec, Boguslaw

    2012-11-13

    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 CO(2). We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O(2) and CO(2) bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO(2) defines an elusive, preactivation complex that contains a metal cation Mg(2+) surrounded by three H(2)O/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.

  2. Comparing GPS, Log, Survey, and Accelerometry to Measure Physical Activity

    PubMed Central

    James, Peter; Weissman, Jennifer; Wolf, Jean; Mumford, Karen; Contant, Cheryl K.; Hwang, Wei-Ting; Taylor, Lynne; Glanz, Karen

    2016-01-01

    Objectives Both self-report and objective measures have strengths and limitations for studying physical activity (PA) and travel. We explored how objectively measured global positioning system (GPS) and accelerometer data matches with travel logs and questionnaires in predicting trip duration and PA. Methods In a study of PA and travel among residents in Atlanta, GA conducted in 2008–2009, 99 participants wore GPS devices and accelerometers, and recorded all trips in a log for 5 consecutive days. Participants also completed a self-administered questionnaire on PA and travel behaviors. Results There was good agreement between GPS and log for assessment of trip duration, although log measures overestimated trip duration (concordance correlation coefficient 0.53 [0.47, 0.59]; Bland-Altman estimate 0.76 [0.16, 3.71] comparing GPS to log). Log measures underestimated light PA and overestimated moderate PA compared to accelerometry when greater than zero moderate PA was reported. Conclusions It is often not feasible to deploy accelerometry or GPS devices in population research because these devices are expensive and require technical expertise and data processing. Questionnaires and logs provide inexpensive tools to assess PA and travel with reasonable concordance with objective measures. However, they have shortcomings in evaluating the presence and amount of light and moderate PA. Future questionnaires and logs should be developed to evaluate sensitivity to light and moderate PA. PMID:26685821

  3. Comparative evaluation of the fate of disinfection byproducts at eight aquifer storage and recovery sites.

    PubMed

    Pavelic, Paul; Dillon, Peter J; Nicholson, Brenton C

    2006-01-15

    Despite the growth in aquifer storage and recovery (ASR) as a technique for the provision of potable water supplies, quantitative data on the fate of disinfection byproducts that may be present in the injected water remain rare. This study evaluates the data from eight ASR sites in Australia and the United States that cover a wide range of source water compositions, hydrogeological environments, and operating conditions. Rates of attenuation and formation of trihalomethanes (THMs) in groundwater were determined using analytical techniques that took dilution effects into account. Half-lives varied by more than 2 orders of magnitude (e.g., <1 to >120 days for total THMs) and were both compound- and site- specific. Chloroform was most persistent, and more highly brominated compounds tended to be less persistent, as has generally been found. For any particular THM compound, much of the variability could be explained by contrasts in geochemical conditions within the aquifer since microbial degradation is the primary mechanism for THM attenuation. As such, bounds on the half-life were defined according to the redox state of the groundwater. In situ formation of some THMs in the aquifer after injection was directly observed at a number of sites, and was predicted to have taken place at all sites. The variance in formation estimates was large between the different methods used. Formation may be more common than previously thought because of the low frequency of groundwater sampling after injection and concomitant attenuation and mixing.

  4. Silver-Coated Nylon Dressing Plus Active DC Microcurrent for Healing of Autogenous Skin Donor Sites

    DTIC Science & Technology

    2013-08-01

    Silver-Coated Nylon Dressing Plus Active DC Microcurrent for Healing of Autogenous Skin Donor Sites Edward W. Malin, MD, Chaya M. Galin, BSN, RN... microcurrent in comparison to silver-coated dressing with sham microcurrent on wound-closure time for autogenous skin donor sites. Methods: Four...hundred five patients were screened for treatment of their donor sites using a silver-coated nylon dressing with either sham or active microcurrent

  5. 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

  6. 76 FR 30696 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-26

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... eligible active uranium and thorium processing site licensees for reimbursement under Title X of the Energy... requires DOE to reimburse eligible uranium and thorium licensees for certain costs of...

  7. 76 FR 24871 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-03

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... from eligible active uranium and thorium processing site licensees for reimbursement under Title X of...). Title X requires DOE to reimburse eligible uranium and thorium licensees for certain costs...

  8. Use of Temperature and Surface Gas Flux as Novel Measures of Microbial Activity at a Crude Oil Spill Site

    NASA Astrophysics Data System (ADS)

    Bekins, B. A.; Warren, E.; Sihota, N. J.; Hostettler, F. D.

    2012-12-01

    Degradation of crude oil in the subsurface has been studied for over 30 years at a spill site located near Bemidji, Minnesota, USA. The well-characterized site is being used to experiment with the use of surface gas flux and temperature measurements as novel methods for quantifying microbial activity. In the largest subsurface oil body, a 2-m-thick smear zone spans the water table 6-8 m below the surface. Methane produced from degradation of the oil diffuses upward and mixes with oxygen from the surface supporting aerobic methanotrophy at 2-4 m depth. The methane oxidation produces CO2 and heat at rates which are hypothetically proportional to other measures of subsurface microbial activity. To test this hypothesis, vertical profiles of temperature and microbial populations, surface CO2 flux, and oil degradation state were measured at three sites in the oil body and one background site. Temperature increases in the oil zone near the water table were 1-4°C above the background site. The site with the highest temperature increase at the water table also had the highest concentrations of gene copy numbers for methanogens (mcrA) and methanotrophs (pmoA) along with the most degraded oil. Surface CO2 flux over the oil sites averaged more than twice that at the background site but was not consistently highest over the site with the highest activity by other measures. One possible explanation for this discrepancy is variation in the effective diffusion coefficient of the vadose zone between the methanotrophic zone and the surface. At the level of the methanotrophic zone, temperatures were elevated 2-6°C over the background values but again the site with greatest average annual temperature increase was not at the most active site. This may be due to enhanced recharge at the most active site, which lies at the center of a local topographic depression where focused recharge occurs. Overall, the temperature and flux data showed significant increases at the oil sites compared

  9. How Do You Like To Learn? Comparing User Preferences and Visit Length of Educational Web Sites.

    ERIC Educational Resources Information Center

    Schaller, David T.; Allison-Bunnell, Steven; Borun, Minda; Chambers, Margaret B.

    This paper reports results of a study designed to determine people's preferences for different types of Web-based educational activity. The primary research question was: How do people's preferences vary among types of Web-based learning activity? Six activity types were identified for comparison: Creative Play, Guided Tour, Interactive Reference,…

  10. A model of the rabies virus glycoprotein active site.

    PubMed

    Rustici, M; Bracci, L; Lozzi, L; Neri, P; Santucci, A; Soldani, P; Spreafico, A; Niccolai, N

    1993-06-01

    The glycoprotein from the neurotropic rabies virus shows a significant homology with the alpha neurotoxin that binds to the nicotinic acetylcholine receptor. The crystal structure of the alpha neurotoxins suggests that the Arg 37 guanidinium group and the Asp 31 side-chain carboxylate of the erabutoxin have stereochemical features resembling those of acetylcholine. Conformational studies on the Asn194-Ser195-Arg196-Gly197 tetrapeptide, an essential part of the binding site of the rabies virus glycoprotein, indicate that the side chains of Asn and Arg could also mimic the acetylcholine structure. This observation is consistent with the recently proposed mechanism of the viral infection.

  11. 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.

  12. Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1

    PubMed Central

    2016-01-01

    Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70–81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1’s lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1’s lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1’s active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar “gating loop” or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates. PMID:28026940

  13. Economic deprivation and racial segregation: comparing Superfund sites in Portland, Oregon and Detroit, Michigan.

    PubMed

    Smith, Chad L

    2009-09-01

    The research presented here weighs the ability of two major explanations of social inequality-Massey and Denton's racial segregation explanation and Wilson's emphasis on economic deprivation (concentrated poverty)-to predict environmental inequality. Two sets of logistic regression analyses are used to predict the location of Superfund sites in Portland, Oregon and Detroit, Michigan providing a conditional understanding of environmental inequality within a larger sociological context. The analysis includes a general examination of the two theories in all census tracts in both cities and a set of analyses focusing upon Black neighborhoods in Detroit. The findings indicate that there is support for explanations of environmental inequality that include both racial segregation and economic deprivation, but that the more powerful of the two is economic deprivation. The results suggest that even though African-American neighborhoods disproportionately house Superfund sites, these facilities are more likely to be located in Black neighborhoods that are economically deprived.

  14. Challenges in Conducting A Multi-Site Randomized Clinical Trial Comparing Treatments for Adolescent Anorexia Nervosa

    PubMed Central

    Brandt, Harry; Woodside, Blake; Agras, Stewart; Halmi, Katherine; Johnson, Craig; Kaye, Walter; Wilfley, Denise

    2011-01-01

    Objective To describe obstacles in the implementation of a controlled treatment trial of adolescent anorexia nervosa (AN). Method The original aim was to enter 240 participants with AN to one of 4 cells: Behavioral family therapy (BFT) plus fluoxetine; BFT plus placebo; systems family therapy (SFT) plus fluoxetine; SFT plus placebo. Results Recruitment was delayed pending a satisfactory resolution concerning participant safety. After 6 months of recruitment it became clear that the medication was associated with poor recruitment leading to a study redesign resulting in a comparison of two types of family therapy with a projected sample size of 160. One site was unable to recruit and was replaced. Discussion Problems with the delineation of safety procedures, recruitment, re-design of the study, and replacement of a site, were the main elements resulting in a 1-year delay. Suggestions are made for overcoming such problems in future AN trials. PMID:21495052

  15. Comparative analyses of spent nuclear fuel transport modal options: Transport options under existing site constraints

    SciTech Connect

    Brentlinger, L.A.; Hofmann, P.L.; Peterson, R.W.

    1989-08-01

    The movement of nuclear waste can be accomplished by various transport modal options involving different types of vehicles, transport casks, transport routes, and intermediate intermodal transfer facilities. A series of systems studies are required to evaluate modal/intermodal spent fuel transportation options in a consistent fashion. This report provides total life-cycle cost and life-cycle dose estimates for a series of transport modal options under existing site constraints. 14 refs., 7 figs., 28 tabs.

  16. Control of Substrate Specificity by Active-Site Residues in Nitrobenzene Dioxygenase

    PubMed Central

    Ju, Kou-San; Parales, Rebecca E.

    2006-01-01

    Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the α subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar Km for the substrate based on product formation rates and whole-cell kinetics. PMID:16517627

  17. Relating subsurface temperature changes to microbial activity at a crude oil-contaminated site

    NASA Astrophysics Data System (ADS)

    Warren, Ean; Bekins, Barbara A.

    2015-11-01

    Crude oil at a spill site near Bemidji, Minnesota has been undergoing aerobic and anaerobic biodegradation for over 30 years, creating a 150-200 m plume of primary and secondary contaminants. Microbial degradation generates heat that should be measurable under the right conditions. To measure this heat, thermistors were installed in wells in the saturated zone and in water-filled monitoring tubes in the unsaturated zone. In the saturated zone, a thermal groundwater plume originates near the residual oil body with temperatures ranging from 2.9 °C above background near the oil to 1.2 °C down gradient. Temperatures in the unsaturated zone above the oil body were up to 2.7 °C more than background temperatures. Previous work at this site has shown that methane produced from biodegradation of the oil migrates upward and is oxidized in a methanotrophic zone midway between the water table and the surface. Enthalpy calculations and observations demonstrate that the temperature increases primarily result from aerobic methane oxidation in the unsaturated zone above the oil. Methane oxidation rates at the site independently estimated from surface CO2 efflux data are comparable to rates estimated from the observed temperature increases. The results indicate that temperature may be useful as a low-cost measure of activity but care is required to account for the correct heat-generating reactions, other heat sources and the effects of focused recharge.

  18. Relating subsurface temperature changes to microbial activity at a crude oil-contaminated site.

    PubMed

    Warren, Ean; Bekins, Barbara A

    2015-11-01

    Crude oil at a spill site near Bemidji, Minnesota has been undergoing aerobic and anaerobic biodegradation for over 30 years, creating a 150-200 m plume of primary and secondary contaminants. Microbial degradation generates heat that should be measurable under the right conditions. To measure this heat, thermistors were installed in wells in the saturated zone and in water-filled monitoring tubes in the unsaturated zone. In the saturated zone, a thermal groundwater plume originates near the residual oil body with temperatures ranging from 2.9°C above background near the oil to 1.2°C down gradient. Temperatures in the unsaturated zone above the oil body were up to 2.7°C more than background temperatures. Previous work at this site has shown that methane produced from biodegradation of the oil migrates upward and is oxidized in a methanotrophic zone midway between the water table and the surface. Enthalpy calculations and observations demonstrate that the temperature increases primarily result from aerobic methane oxidation in the unsaturated zone above the oil. Methane oxidation rates at the site independently estimated from surface CO2 efflux data are comparable to rates estimated from the observed temperature increases. The results indicate that temperature may be useful as a low-cost measure of activity but care is required to account for the correct heat-generating reactions, other heat sources and the effects of focused recharge.

  19. Relating subsurface temperature changes to microbial activity at a crude oil-contaminated site

    USGS Publications Warehouse

    Warren, Ean; Bekins, Barbara A.

    2015-01-01

    Crude oil at a spill site near Bemidji, Minnesota has been undergoing aerobic and anaerobic biodegradation for over 30 years, creating a 150–200 m plume of primary and secondary contaminants. Microbial degradation generates heat that should be measurable under the right conditions. To measure this heat, thermistors were installed in wells in the saturated zone and in water-filled monitoring tubes in the unsaturated zone. In the saturated zone, a thermal groundwater plume originates near the residual oil body with temperatures ranging from 2.9 °C above background near the oil to 1.2 °C down gradient. Temperatures in the unsaturated zone above the oil body were up to 2.7 °C more than background temperatures. Previous work at this site has shown that methane produced from biodegradation of the oil migrates upward and is oxidized in a methanotrophic zone midway between the water table and the surface. Enthalpy calculations and observations demonstrate that the temperature increases primarily result from aerobic methane oxidation in the unsaturated zone above the oil. Methane oxidation rates at the site independently estimated from surface CO2 efflux data are comparable to rates estimated from the observed temperature increases. The results indicate that temperature may be useful as a low-cost measure of activity but care is required to account for the correct heat-generating reactions, other heat sources and the effects of focused recharge.

  20. The oxidation state of active site thiols determines activity of saccharopine dehydrogenase at low pH.

    PubMed

    Bobyk, Kostyantyn D; Kim, Sang Gon; Kumar, Vidya Prasanna; Kim, Sung-Kun; West, Ann H; Cook, Paul F

    2011-09-15

    Saccharopine dehydrogenase catalyzes the NAD-dependent conversion of saccharopine to generate L-lysine and α-ketoglutarate. A disulfide bond between cysteine 205 and cysteine 249, in the vicinity of the dinucleotide-binding site, is observed in structures of the apoenzyme, while a dithiol is observed in a structure with AMP bound, suggesting preferential binding of the dinucleotide to reduced enzyme. Mutation of C205 to S gave increased values of V/E(t) and V/KE(t) at pH 7 compared to wild type. Primary deuterium and solvent deuterium kinetic isotope effects suggest the catalytic pathway, which includes the hydride transfer and hydrolysis steps, contributes more to rate limitation in C205S, but the rates of the two steps relative to one another remain the same. There is a large increase in the rate constants V₁/E(t) and V₁/K(NAD)Et at pH values below 7 compared to WT. Data indicate the low pH increase in activity results from a decreased sensitivity of the C205S mutant enzyme to the protonation state of an enzyme group with a pK(a) of about 7, likely responsible for a pH-dependent conformational change. Reduction of WT and C205S mutant enzymes with TCEP gives equal activities at pH 6, consistent with the increased activity observed for the C205S mutant enzyme.

  1. 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.

  2. All the catalytic active sites of MoS2 for hydrogen evolution

    DOE PAGES

    Li, Guoqing; Zhang, Du; Qiao, Qiao; ...

    2016-11-29

    MoS2 presents a promising low-cost catalyst for the hydrogen evolution reaction (HER), but the understanding about its active sites has remained limited. Here we present an unambiguous study of the catalytic activities of all possible reaction sites of MoS2, including edge sites, sulfur vacancies, and grain boundaries. We demonstrate that, in addition to the well-known catalytically active edge sites, sulfur vacancies provide another major active site for the HER, while the catalytic activity of grain boundaries is much weaker. Here, the intrinsic turnover frequencies (Tafel slopes) of the edge sites, sulfur vacancies, and grain boundaries are estimated to be 7.5more » s–1 (65–75 mV/dec), 3.2 s–1 (65–85 mV/dec), and 0.1 s–1 (120–160 mV/dec), respectively. We also demonstrate that the catalytic activity of sulfur vacancies strongly depends on the density of the vacancies and the local crystalline structure in proximity to the vacancies. Unlike edge sites, whose catalytic activity linearly depends on the length, sulfur vacancies show optimal catalytic activities when the vacancy density is in the range of 7–10%, and the number of sulfur vacancies in high crystalline quality MoS2 is higher than that in low crystalline quality MoS2, which may be related with the proximity of different local crystalline structures to the vacancies.« less

  3. Elevated Ground Temperatures at Crude Oil Spill Sites due to Microbial Activity

    NASA Astrophysics Data System (ADS)

    Warren, E.; Bekins, B. A.

    2009-12-01

    Crude oil near the water table at spill sites near Bemidji and Cass Lake, Minnesota, has been undergoing aerobic and anaerobic biodegradation for decades. Because the reactions are exothermic, biodegradation of oil compounds will produce measurable temperature increases if heat is generated faster than it is transported away from the oil body. Subsurface temperatures at the two spill sites were measured with thermistors at multiple depths in groundwater monitoring wells and water-filled tubes in the vadose zone. Temperatures in selected wells were measured in the summer of 2007, 2008, and 2009. At the Bemidji site, temperatures measured in the summer ranged from a low of 6.3 oC in the background well to a high of 9.2 oC within wells in the oil-contaminated zone. From year to year, background minimum temperatures were constant within +/- 0.05 oC while maximum temperatures within the oil-contaminated zone remained within +/- 0.25 oC. Seasonal changes in temperature in the plume as measured by data loggers exceeded 4 oC, which was far greater than the year to year change in the summer measurements. Seasonal variability was greater near the water table than at depth. It is unclear whether this variability is due to subsurface hydrology or microbial activity. Temperatures in the vadose zone were warmer near and down-gradient from the oil body compared to the background indicating the heat from the oil and plume propagates up and outward into the vadose zone. At the Cass Lake site, summer temperatures in 2009 were 6.4 oC in the background and 11.5 oC in wells near the oil. Reaction rates inferred from chemical data were compared to heating required in a 3-dimension energy transport model of the subsurface. The increased temperature compared well to the expected heat production from biodegradation reactions occurring in the oil and plume. Results indicate that microbial activity in sediments contaminated with crude oil undergoing biodegradation can be detected using

  4. 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

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

    ERIC Educational Resources Information Center

    Pauls, John

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

  6. Comparative assessment of five potential sites for hydrothermal magma systems: geochemistry

    SciTech Connect

    White, A.F.

    1980-08-01

    A brief discussion is given of the geochemical objectives and questions that must be addressed in such an evaluation. A summary of the currently published literature that is pertinent in answering these questions is presented for each of the five areas: The Geysers-Clear Lake region, Long Valley, Rio Grand Rift, Roosevelt Hot Springs, and the Salton Trough. The major geochemical processes associated with proposed hydrothermal sites are categorized into three groups for presentation: geochemistry of magma and associated volcanic rocks, geochemistry of hydrothermal solutions, and geochemistry of hydrothermal alteration. (MHR)

  7. An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus.

    PubMed

    Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

    2016-10-01

    Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

  8. 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

  9. Catalytic and Structural Role of a Conserved Active Site Histidine in Berberine Bridge Enzyme

    PubMed Central

    2012-01-01

    Berberine bridge enzyme (BBE) is a paradigm for the class of bicovalently flavinylated oxidases, which catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine. His174 was identified as an important active site residue because of its role in the stabilization of the reduced state of the flavin cofactor. It is also strictly conserved in the family of BBE-like oxidases. Here, we present a detailed biochemical and structural characterization of a His174Ala variant supporting its importance during catalysis and for the structural organization of the active site. Substantial changes in all kinetic parameters and a decrease in midpoint potential were observed for the BBE His174Ala variant protein. Moreover, the crystal structure of the BBE His174Ala variant showed significant structural rearrangements compared to wild-type enzyme. On the basis of our findings, we propose that His174 is part of a hydrogen bonding network that stabilizes the negative charge at the N1–C2=O locus via interaction with the hydroxyl group at C2′ of the ribityl side chain of the flavin cofactor. Hence, replacement of this residue with alanine reduces the stabilizing effect for the transiently formed negative charge and results in drastically decreased kinetic parameters as well as a lower midpoint redox potential. PMID:22757961

  10. Observing the formation of ice and organic crystals in active sites.

    PubMed

    Campbell, James M; Meldrum, Fiona C; Christenson, Hugo K

    2017-01-31

    Heterogeneous nucleation is vital to a wide range of areas as diverse as ice nucleation on atmospheric aerosols and the fabrication of high-performance thin films. There is excellent evidence that surface topography is a key factor in directing crystallization in real systems; however, the mechanisms by which nanoscale pits and pores promote nucleation remain unclear. Here, we use natural cleavage defects on Muscovite mica to investigate the activity of topographical features in the nucleation from vapor of ice and various organic crystals. Direct observation of crystallization within surface pockets using optical microscopy and also interferometry demonstrates that these sharply acute features provide extremely effective nucleation sites and allows us to determine the mechanism by which this occurs. A confined phase is first seen to form along the apex of the wedge and then grows out of the pocket opening to generate a bulk crystal after a threshold saturation has been achieved. Ice nucleation proceeds in a comparable manner, although our resolution is insufficient to directly observe a condensate before the growth of a bulk crystal. These results provide insight into the mechanism of crystal deposition from vapor on real surfaces, where this will ultimately enable us to use topography to control crystal deposition on surfaces. They are also particularly relevant to our understanding of processes such as cirrus cloud formation, where such topographical features are likely candidates for the "active sites" that make clay particles effective nucleants for ice in the atmosphere.

  11. Distance between two active-site lysines of ribulosebis-phosphate carboxylase/oxygenase

    SciTech Connect

    Lee, E.H.; Hartman, F.C.

    1986-05-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of the title enzyme (Lys-166 and Lys-329 in the Rhodospirillum rubrum enzyme and Lys-175 and Lys-334 in the spinach enzyme). Because the two lysines are mutually exclusive to various reagents, they appear to be in proximity. To challenge this postulate, the authors have explored the reactions of the R. rubrum enzyme (a homodimer) with chemical cross-linking agents. 4,4'-Diisothiocyano-2,2'-disulfonate stilbene, which spans 12 A, rapidly inactivates the enzyme with protection afforded by the competitive inhibitor 2-carboxyribitol-1,5-bisphosphate. The inactivated enzyme was subjected to gel filtration in the presence of urea to remove material arising from intersubunit or intermolecular cross-linking. The monomeric fraction was digested with trypsin; inspection of the digest by HPLC revealed that over-half of the incorporated reagent was associated with a single peptide. This peptide was purified by successive ion-exchange chromatography and gel filtration. The amino acid composition and sequence of the purified peptide demonstrated that it is comprised of two chains, encompassing position 149-168 and 314-337 of the original protein subunit and connected by a cross-link between Lys-166 and Lys-329. Thus, the two active-site lysines can be juxtaposed only 12 A apart.

  12. Selectivity of fungal sesquiterpene synthases: role of the active site's H-1 alpha loop in catalysis.

    PubMed

    López-Gallego, Fernando; Wawrzyn, Grayson T; Schmidt-Dannert, Claudia

    2010-12-01

    Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-α1 loop in catalysis in three previously characterized fungal sesquiterpene synthases. The H-α1 loops of Cop3, Cop4, and Cop6 from Coprinus cinereus were altered by site-directed mutagenesis and the resultant product profiles were analyzed by gas chromatography-mass spectrometry and compared to the wild-type enzymes. In addition, we examine the effect of swapping the H-α1 loop from the promiscuous enzyme Cop4 with the more selective Cop6 and the effect of acidic or basic conditions on loop mutations in Cop4. Directed mutations of the H-α1 loop had a marked effect on the product profile of Cop3 and Cop4, while little to no change was shown in Cop6. Swapping of the Cop4 and Cop6 loops with one another was again shown to influence the product profile of Cop4, while the product profile of Cop6 remained identical to the wild-type enzyme. The loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. These results affirm the role of the H-α1 loop in catalysis and provide a potential target to increase the product diversity of terpene synthases.

  13. 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.

  14. Differential response of chloride binding sites to elevated temperature: a comparative study in spinach thylakoids and PSII-enriched membranes.

    PubMed

    Tiwari, Arjun; Jajoo, Anjana; Bharti, Sudhakar; Mohanty, Prasanna

    2007-01-01

    A study of heat effects was performed in thylakoids and photosystem II (PSII)-enriched membranes isolated from spinach in relation to Cl(-)-induced activation of PSII catalyzed oxygen evolution and the retention of Cl(-) in the PSII complex. For this, Cl(-)-sufficient membranes and low-Cl(-) membranes were used. The presence of Cl(-) in the reaction medium did accelerate oxygen evolution, which remained unaffected by heat treatment up to 40 degrees C in PSII membranes and up to 42.5 degrees C in thylakoids. Heat resistance of Cl(-)-induced activation of oxygen evolution was found to be independent of the presence of 'bound Cl(-)' in the preparations. However, the functional stability of the PSII complex during heat treatment showed a marked dependence on the presence of bound Cl(-) in PSII. Electron paramagnetic resonance study of manganese (Mn) release per reaction center/Y (D) (+) showed that there was little loss of Mn(2+) up to 42 degrees C in our preparations, although the PSII activity was significantly lowered. These observations together with data from steady state chlorophyll a fluorescence imply that the site of action of Cl(-) causing direct activation of oxygen evolution was different from the site of primary heat damage. A differential response of chloride binding sites to heat stress was observed. The high-affinity (tightly bound, slow exchanging) site of chloride is affected earlier ( approximately 37 degrees C) while low-affinity (loosely bound, fast exchanging) site gets affected at higher temperatures (42.5 degrees C in thylakoids and 40 degrees C in the case of PSII-enriched membranes).

  15. 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.

  16. Activation of brown adipose tissue mitochondrial GDP binding sites

    SciTech Connect

    Swick, A.G.

    1987-01-01

    The primary function of brown adipose tissue (BAT) is heat production. This ability is attributed to the existence of a unique inner mitochondrial membrane protein termed the uncoupling protein or thermogenin. This protein is permeable to H+ and thus allows respiration (and therefore thermogenesis) to proceed at a rapid rate, independent of ADP phosphorylation. Proton conductance can be inhibited by the binding of purine nucleotides to the uncoupling protein. The binding of (/sup 3/H)-GDP to BAT mitochondria is frequently used as a measure of BAT thermogenic activity. Rats fed a diet that was low but adequate in protein exhibited a decrease in feed efficiency. In addition, BAT thermogenesis was activated as indicated by an elevation in the level of GDP binding to BAT mitochondria. This phenomena occurred in older rats and persisted over time.

  17. Structure and Reactivity of the Phosphotriesterase Active Site

    DTIC Science & Technology

    2002-01-01

    characterize different catalytic conformations for chorismate mutase . Preliminary evidence for water binding in phosphotriesterase suggests that activity in...MD/QM study of the chorismate mutase catalyzed Claisen rearrangement reaction. 2001.subm. J.Phys.Chem.B 22.Day, P.N.J., J.H.; Gordon,M.S.; Webb,S.P...Claisen rearrangement of an unusual substrate in chorismate mutase . 2001.subm. J.Phys.Chem.B 38.Stevens, W.J., Basch,H., Krauss,M., Compact effective

  18. Phase I and II liver enzyme activities in juvenile alligators (Alligator mississippiensis) collected from three sites in the Kissimmee-Everglades drainage, Florida (USA).

    PubMed

    Gunderson, M P; Oberdörster, E; Guillette, L J

    2004-10-01

    We examined CYP1A (measured using hepatic EROD and MROD activities) and glutathione-S-transferase (GST) activities in juvenile alligators (Alligator mississippiensis) collected from three sites with varying contamination in the Kissimmee-Everglades drainage in south Florida. We hypothesized that contaminants present in areas with intermediate or higher contaminant concentrations would alter hepatic enzyme activities in juvenile alligators from those sites when compared to hepatic enzyme activity in animals from the area with the least contamination. EROD activity was found to be higher in animals from the site with lower reported levels of contamination relative to those from the site with the highest reported contamination suggesting an inhibition of CYP1A expression or activity. No differences among animals from the three sites were observed for hepatic MROD and GST activities. A significant negative relationship between EROD, MROD, and GST activities and body size was exhibited in alligators from the site with the lowest contamination. No relationship between body size and hepatic enzyme activity was found in animals from the sites with intermediate and higher contamination, suggesting that contaminants present at these sites act to alter this relationship. No correlation was observed in this study between plasma steroid concentrations (estradiol-17 beta or testosterone) and hepatic EROD, MROD, or GST activities.

  19. Trace metal biogeochemistry in mangrove ecosystems: a comparative assessment of acidified (by acid sulfate soils) and non-acidified sites.

    PubMed

    Nath, Bibhash; Birch, Gavin; Chaudhuri, Punarbasu

    2013-10-01

    The generation of acidity and subsequent mobilization of toxic metals induced by acid sulfate soils (ASSs) are known to cause severe environmental damage to many coastal wetlands and estuaries of Australia and worldwide. Mangrove ecosystems serve to protect coastal environments, but are increasingly threatened from such ASS-induced acidification due to variable hydrological conditions (i.e., inundation-desiccation cycles). However, the impact of such behaviors on trace metal distribution, bio-availability and accumulation in mangrove tissues, i.e., leaves and pneumatophores, are largely unknown. In this study, we examined how ASS-induced acidifications controlled trace metal distribution and bio-availability in gray mangrove (Avicennia marina) soils and in tissues in the Kooragang wetland, New South Wales, Australia. We collected mangrove soils, leaves and pneumatophores from a part of the wetland acidified from ASS (i.e., an affected site) for detailed biogeochemical studies. The results were compared with samples collected from a natural intertidal mangrove forest (i.e., a control site) located within the same wetland. Soil pH (mean: 5.90) indicated acidic conditions in the affected site, whereas pH was near-neutral (mean: 7.17) in the control site. The results did not show statistically significant differences in near-total and bio-available metal concentrations, except for Fe and Mn, between affected and control sites. Iron concentrations were significantly (p values≤0.001) greater in the affected site, whereas Mn concentrations were significantly (p values≤0.001) greater in the control site. However, large proportions of near-total metals were potentially bio-available in control sites. Concentrations of Fe and Ni were significantly (p values≤0.001) greater in leaves and pneumatophores of the affected sites, whereas Mn, Cu, Pb and Zn were greater in control sites. The degree of metal bio-accumulation in leaves and pneumatophores suggest contrasting

  20. Comparative Evaluation of Drug Deposition in Hair Samples Collected from Different Anatomical Body Sites.

    PubMed

    Tzatzarakis, Manolis N; Alegakis, Athanasios K; Kavvalakis, Matthaios P; Vakonaki, Elena; Stivaktakis, Polychronis D; Kanaki, Katerina; Vardavas, Alexander I; Barbounis, Emmanouil G; Tsatsakis, Aristidis M

    2016-12-15

    In this study, we focused on the validation of a method for the simultaneous detection and quantification of cannabinoids, cocaine and opiates in hair as well as on the distribution of the drugs deposition in hair collected from different anatomical body sites. The proposed analytical procedure was validated for various parameters such as selectivity, linearity, limit of quantification, precision, accuracy, matrix effect and recovery. Four hundred and eighty-one samples were collected during 2010-2015 from 231 drug abusers. A 6-h ultrasonic-assisted methanolic extraction was applied for the isolation of the drugs. The analysis was performed in an liquid chromatography-mass spectrometry system for the opiates and cocaine and in a gas chromatography-mass spectrometry system for the cannabinoids. Cocaine was the most frequent detected drug (68.8-80.5%) followed by cannabinoids (47.6-63.3%) and opiates (34.7-46.7%) depending on the body site that the samples were collected. The mean concentrations of Δ(9)-tetrahydrocannabinol (THC) were 0.63 ± 2.11 for head, 0.54 ± 1.03 for pubic, 0.34 ± 0.51 for axillary and 0.18 ± 0.18 ng/mg for chest hair samples. The values of cocaine were 6.52 ± 15.98, 4.64 ± 10.77, 6.96 ± 38.21 and 3.94 ± 6.35 ng/mg, while the values of 6-monoacetylmorphine (MAM) were 3.33 ± 5.89, 3.06 ± 9.33, 1.37 ± 1.37 and 16.4 ± 1.77 ng/mg for head, pubic, axillary and chest samples, respectively. Differences between the detected concentrations of cocaine and opiates between the hair samples of different anatomical sites, as well as the ratio of drug metabolites to the parent compounds were observed in some cases. Statistically significant differences in the mean detected levels were noticed for morphine and heroin between head and pubic hair and also for cocaine and benzoylecgonine, between head and axillary hair samples. Moreover, the ratio of MAM to morphine and THC to cannabinol seems to correlate statistically with the total opiate or

  1. Anesthetic Activity of Alfaxalone Compared with Ketamine in Mice.

    PubMed

    Siriarchavatana, Parkpoom; Ayers, Jessica D; Kendall, Lon V

    2016-01-01

    Alfaxalone encased in hydroxypropyl-β -cyclodextrin is a neuroactive steroid compound that has recently been approved in the United States for use as an anesthetic in dogs and cats. We evaluated the use of alfaxalone compared with ketamine, both alone and in combination with xylazine, for anesthesia of C57BL/6 mice. We assessed time to onset of anesthesia, duration of action, reflex responses, respiratory rate, and clinical signs. Alfaxalone (80 mg/kg IP) induced a light surgical plane of anesthesia in all mice, with a time to onset of 2.2 ± 0.2 min and duration of 57.1 ± 3.8 min, whereas ketamine (80 mg/kg IP) provided only sedative effects (time to onset, 5.4 ± 0.4 min; duration, 6.9 ± 0.8 min). Clinically, alfaxalone caused a spectrum of activities, including popcorn-like jumping movements after injection, intense scratching of the face, hyperresponsiveness to noise or touch, and marked limb jerking during recovery. Adding xylazine to the single-agent protocols achieved deep surgical anesthesia (duration: alfaxalone + xylazine, 80.3 ± 17.8 min; ketamine + xylazine, 37.4 ± 8.2 min) and ameliorated the adverse clinical signs. Our preliminary analysis suggests that, because of its side effects, alfaxalone alone is not a viable anesthetic option for mice. Although alfaxalone combined with xylazine appeared to be a more viable option, some mice still experienced mild adverse reactions, and the long duration of action might be problematic regarding the maintenance of body temperature and monitoring of recovery. Further studies evaluating different routes of administration and drug combinations are warranted.

  2. Pathways of H2 toward the Active Site of [NiFe]-Hydrogenase

    PubMed Central

    Teixeira, Vitor H.; Baptista, António M.; Soares, Cláudio M.

    2006-01-01

    Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H2), but little is known about the diffusion of H2 toward the active site. Here we analyze pathways for H2 permeation using molecular dynamics (MD) simulations in explicit solvent. Various MD simulation replicates were done, to improve the sampling of the system states. H2 easily permeates hydrogenase in every simulation and it moves preferentially in channels. All H2 molecules that reach the active site made their approach from the side of the Ni ion. H2 is able to reach distances of <4 Å from the active site, although after 6 Å permeation is difficult. In this region we mutated Val-67 into alanine and perform new MD simulations. These simulations show an increase of H2 inside the protein and at lower distances from the active site. This valine can be a control point in the H2 access to the active center. PMID:16731562

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

    PubMed

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

    2006-03-01

    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.

  4. Research in Support of Remediation Activities at the Savannah River Site

    SciTech Connect

    Seaman, J.C., B.B. Looney and M.K. Harris

    2007-01-01

    The USDOE Savannah River Site (SRS), an 803-km{sup 2} (310-mile{sup 2}) facility located south of Aiken, SC on the upper Atlantic Coastal Plain and bounded to the west by the Savannah River, was established in the 1950s for the production and refinement of nuclear materials. To fulfill this mission during the past 50 years SRS has operated five nuclear reactors, two large chemical separation areas, waste disposal facilities (landfills, waste ponds, waste tanks, and waste stabilization), and a large number of research and logistics support facilities. Contaminants of concern (COC) resulting from site operations include chlorinated solvents, radionuclides, metals, and metalloids, often found as complex mixtures that greatly complicate remediation efforts when compared with civilian industries. The objective of this article is to provide a description of the lithology and hydrostratigraphy of the SRS, as well as a brief history of site operations and research activities as a preface to the current special section of Vadose Zone Journal (VZJ) dedicated to SRS, focusing mainly on issues that are unique to the USDOE complex. Contributions to the special section reflect a diverse range of topics, from hydrologic tracer experiments conducted both within the vadose and saturated zones to studies specifically aimed at identifying geochemical processes controlling the migration and partitioning of specific contaminants (e.g., TCE, {sup 137}Cs, U, and Pu) in SRS subsurface environments. Addressing the diverse environmental challenges of the SRS provides a unique opportunity to conduct both fundamental and applied research across a range of experimental scales. Hence, the SRS has been a pioneering force in several areas of environmental research and remediation, often through active interdisciplinary collaboration with researchers from other USDOE facilities, academic and federal institutions, and commercial entities.

  5. A Processive Carbohydrate Polymerase That Mediates Bifunctional Catalysis Using a Single Active Site

    PubMed Central

    May, John F.; Levengood, Matthew R.; Splain, Rebecca A.; Brown, Christopher D.; Kiessling, Laura L.

    2012-01-01

    Even in the absence of a template, glycosyltransferases can catalyze the synthesis of carbohydrate polymers of specific sequence. The paradigm has been that one enzyme catalyzes the formation of one type of glycosidic linkage, yet certain glycosyltransferases generate polysaccharide sequences composed of two distinct linkage types. In principle, bifunctional glycosyltransferases can possess separate active sites for each catalytic activity or one active site with dual activities. We encountered the fundamental question of one or two distinct active sites in our investigation of the galactosyltransferase GlfT2. GlfT2 catalyzes the formation of mycobacterial galactan, a critical cell-wall polymer composed of galactofuranose residues connected with alternating, regioisomeric linkages. We found that GlfT2 mediates galactan polymerization using only one active site that manifests dual regioselectivity. Structural modeling of the bifunctional glycosyltransferases hyaluronan synthase and cellulose synthase suggests that these enzymes also generate multiple glycosidic linkages using a single active site. These results highlight the versatility of glycosyltransferases for generating polysaccharides of specific sequence. We postulate that a hallmark of processive elongation of a carbohydrate polymer by a bifunctional enzyme is that one active site can give rise to two separate types of glycosidic bonds. PMID:22217153

  6. Seven-day activity and self-report compared to a direct measure of physical activity.

    PubMed

    Taylor, C B; Coffey, T; Berra, K; Iaffaldano, R; Casey, K; Haskell, W L

    1984-12-01

    To determine how well a seven-day interview-administered activity recall used in a large epidemiologic study at Stanford University reflected seven days of self-reported activity and directly measured physical activity, 30 white males, mean age 52 years, recorded daily physical activity for a week, and half of these wore an ambulatory solid-state minicomputer (Vitalog) which measures continuous heart rate and motion. Total hours of moderate, hard, and hard plus very hard activity were not significantly different for weekdays and weekends for self-report and recall and were significantly correlated. Total energy expenditure for subjects wearing the Vitalog averaged 38.5 +/- 6.7 kcal/kg/day compared to an average of 37.7 +/- 4.5 kcal/kg/day for recall or 39.6 +/- 7.2 kcal/kg/day for self-report. Conditioning activities are best remembered followed by home or leisure and job activities. Mean hours of sleep per week night were significantly greater reported by self-report than reported by recall, but the two were significantly correlated. It is concluded that a seven-day activity recall accurately reflects mean kcal/day expenditure, with conditioning activities being the best recalled. A self-report log used in conjunction with an interview-based seven-day recall might maximize accuracy of recall.

  7. Assessing the use of existing data to compare plains fish assemblages collected from random and fixed sites in Colorado

    USGS Publications Warehouse

    Zuellig, Robert E.; Crockett, Harry J.

    2013-01-01

    The U.S. Geological Survey, in cooperation with Colorado Parks and Wildlife, assessed the potential use of combining recently (2007 to 2010) and formerly (1992 to 1996) collected data to compare plains fish assemblages sampled from random and fixed sites located in the South Platte and Arkansas River Basins in Colorado. The first step was to determine if fish assemblages collected between 1992 and 1996 were comparable to samples collected at the same sites between 2007 and 2010. If samples from the two time periods were comparable, then it was considered reasonable that the combined time-period data could be used to make comparisons between random and fixed sites. In contrast, if differences were found between the two time periods, then it was considered unreasonable to use these data to make comparisons between random and fixed sites. One-hundred samples collected during the 1990s and 2000s from 50 sites dispersed among 19 streams in both basins were compiled from a database maintained by Colorado Parks and Wildlife. Nonparametric multivariate two-way analysis of similarities was used to test for fish-assemblage differences between time periods while accounting for stream-to-stream differences. Results indicated relatively weak but significant time-period differences in fish assemblages. Weak time-period differences in this case possibly were related to changes in fish assemblages associated with environmental factors; however, it is difficult to separate other possible explanations such as limited replication of paired time-period samples in many of the streams or perhaps differences in sampling efficiency and effort between the time periods. Regardless, using the 1990s data to fill data gaps to compare random and fixed-site fish-assemblage data is ill advised based on the significant separation in fish assemblages between time periods and the inability to determine conclusive explanations for these results. These findings indicated that additional sampling will

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

    PubMed Central

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

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

  9. Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site

    PubMed Central

    Águila, Sonia; Teruel-Montoya, Raúl; Vicente, Vicente; Corral, Javier; Martínez-Martínez, Irene

    2016-01-01

    Heparanase is an endoglycosidase that participates in morphogenesis, tissue repair, heparan sulphates turnover and immune response processes. It is over-expressed in tumor cells favoring the metastasis as it penetrates the endothelial layer that lines blood vessels and facilitates the metastasis by degradation of heparan sulphate proteoglycans of the extracellular matrix. Heparanase may also affect the hemostatic system in a non-enzymatic manner, up-regulating the expression of tissue factor, which is the initiator of blood coagulation, and dissociating tissue factor pathway inhibitor on the cell surface membrane of endothelial and tumor cells, thus resulting in a procoagulant state. Trying to check the effect of heparanase on heparin, a highly sulphated glycosaminoglycan, when it activates antithrombin, our results demonstrated that heparanase, but not proheparanase, interacted directly with antithrombin in a non-covalent manner. This interaction resulted in the activation of antithrombin, which is the most important endogenous anticoagulant. This activation mainly accelerated FXa inhibition, supporting an allosteric activation effect. Heparanase bound to the heparin binding site of antithrombin as the activation of Pro41Leu, Arg47Cys, Lys114Ala and Lys125Alaantithrombin mutants was impaired when it was compared to wild type antithrombin. Intrinsic fluorescence analysis showed that heparanase induced an activating conformational change in antithrombin similar to that induced by heparin and with a KD of 18.81 pM. In conclusion, under physiological pH and low levels of tissue factor, heparanase may exert a non-enzymatic function interacting and activating the inhibitory function of antithrombin. PMID:27322195

  10. Preliminary assessment of several parameters to measure and compare usefulness of the CEOS reference pseudo-invariant calibration sites

    USGS Publications Warehouse

    Chander, Gyanesh; Angal, Amit; Xiong, Xiaoxiong; Helder, Dennis L.; Mishra, Nischal; Choi, Taeyoung; Wu, Aisheng

    2010-01-01

    Test sites are central to any future quality assurance and quality control (QA/QC) strategy. The Committee on Earth Observation Satellites (CEOS) Working Group for Calibration and Validation (WGCV) Infrared Visible Optical Sensors (IVOS) worked with collaborators around the world to establish a core set of CEOS-endorsed, globally distributed, reference standard test sites (both instrumented and pseudo-invariant) for the post-launch calibration of space-based optical imaging sensors. The pseudo-invariant calibration sites (PICS) have high reflectance and are usually made up of sand dunes with low aerosol loading and practically no vegetation. The goal of this paper is to provide preliminary assessment of "several parameters" than can be used on an operational basis to compare and measure usefulness of reference sites all over the world. The data from Landsat 7 (L7) Enhanced Thematic Mapper Plus (ETM+) and the Earth Observing-1 (EO-1) Hyperion sensors over the CEOS PICS were used to perform a preliminary assessment of several parameters, such as usable area, data availability, top-of-atmosphere (TOA) reflectance, at-sensor brightness temperature, spatial uniformity, temporal stability, spectral stability, and typical spectrum observed over the sites.

  11. High and dry? Comparing active dry EEG electrodes to active and passive wet electrodes.

    PubMed

    Mathewson, Kyle E; Harrison, Tyler J L; Kizuk, Sayeed A D

    2017-01-01

    Dry electrodes are becoming popular for both lab-based and consumer-level electrophysiological-recording technologies because they better afford the ability to move traditional lab-based research into the real world. It is unclear, however, how dry electrodes compare in data quality to traditional electrodes. The current study compared three EEG electrode types: (a) passive-wet electrodes with no onboard amplification, (b) actively amplified, wet electrodes with moderate impedance levels, and low impedance levels, and (c) active-dry electrodes with very high impedance. Participants completed a classic P3 auditory oddball task to elicit characteristic EEG signatures and event-related potentials (ERPs). Across the three electrode types, we compared single-trial noise, average ERPs, scalp topographies, ERP noise, and ERP statistical power as a function of number of trials. We extended past work showing active electrodes' insensitivity to moderate levels of interelectrode impedance when compared to passive electrodes in the same amplifier. Importantly, the new dry electrode system could reliably measure EEG spectra and ERP components comparable to traditional electrode types. As expected, however, dry active electrodes with very high interelectrode impedance exhibited marked increases in single-trial and average noise levels, which decreased statistical power, requiring more trials to detect significant effects. This power decrease must be considered as a trade-off with the ease of application and long-term use. The current results help set constraints on experimental design with novel dry electrodes, and provide important evidence needed to measure brain activity in novel settings and situations.

  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. 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

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... uranium and thorium processing site licensees for reimbursement under Title X of the Energy Policy Act of... requires DOE to reimburse eligible uranium and thorium licensees for certain costs of...

  14. Comparative activity of Choristoneura fumiferana nucleopolyhedrovirus propagated in different hosts.

    PubMed

    Ebling, Peter M

    2004-07-01

    The biological activity of the Ireland strain of Choristoneura fumiferana (Clem) nucleopolyhedrovirus (CfMNPV) propagated in different hosts was determined to provide the basis upon which genetically modified CfMNPV, or other naturally occurring isolates, should be compared. Occlusion bodies (OB) derived from CF-203 cells were significantly larger and more pathogenic than those propagated in vivo when tested against the fifth larval instar of C fumiferana (Clem) and C occidentalis Freeman. The dose-responses (LD50 and LD95, expressed as occlusion bodies per larva) of C fumiferana larvae to in vitro-propagated OBs were 274 and 5785, respectively. The values of LD50 and LD95 to C occidentalis larvae were 19 and 118, respectively. There were no significant differences in pathogenicity or size when OBs propagated in C fumiferana larvae were tested against either insect species, nor were there significant differences for OBs propagated in C occidentalis larvae. The LD50 and LD95 of in vivo-produced OBs to C fumiferana were 925 and 61988, respectively. The LD50 and LD95 to C occidentalis were 50 and 453, respectively. OBs propagated in vitro had a mean volume of 13.13 microm3, whereas those propagated in vivo ranged from 0.84 to 1.41 microm3. The median survival time-responses (ST50) of fifth-instar C fumiferana or C occidentalis larvae to OBs propagated in vivo were not significantly different from those propagated in vitro at the dosage levels tested. Values of ST50 of C fumiferana larvae to in vitro- and in vivo-produced OBs at dosages causing less than 50% mortality rangedfrom 9.6 to 9.8 days post-inoculation (dpi), whereas a LD95 dose resulted in ST50 values ranging from 7.3 to 7.7 days. ST50 values of C occidentalis larvae at dosages causing less than 50% mortality ranged from 9.8 to 10.2 dpi, whereas a LD95 dose resulted in ST50 values ranging from 9.5 to 9.8 dpi. The median feeding cessation time-response (FT50) of fifth-instar C fumiferana larvae to OBs

  15. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...). (e) For all asbestos-containing waste material received, the owner or operator of the active waste... 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...

  16. Comparative analysis of contextual bias around the translation initiation sites in plant genomes.

    PubMed

    Gupta, Paras; Rangan, Latha; Ramesh, T Venkata; Gupta, Mudit

    2016-09-07

    Nucleotide distribution around translation initiation site (TIS) is thought to play an important role in determining translation efficiency. Kozak in vertebrates and later Joshi et al. in plants identified context sequence having a key role in translation efficiency, but a great variation regarding this context sequence has been observed among different taxa. The present study aims to refine the context sequence around initiation codon in plants and addresses the sampling error problem by using complete genomes of 7 monocots and 7 dicots separately. Besides positions -3 and +4, significant conservation at -2 and +5 positions was also found and nucleotide bias at the latter two positions was shown to directly influence translation efficiency in the taxon studied. About 1.8% (monocots) and 2.4% (dicots) of the total sequences fit the context sequence from positions -3 to +5, which might be indicative of lower number of housekeeping genes in the transcriptome. A three base periodicity was observed in 5' UTR and CDS of monocots and only in CDS of dicots as confirmed against random occurrence and annotation errors. Deterministic enrichment of GCNAUGGC in monocots, AANAUGGC in dicots and GCNAUGGC in plants around TIS was also established (where AUG denotes the start codon), which can serve as an arbiter of putative TIS with efficient translation in plants.

  17. Identification of active sites in amidase: Evolutionary relationship between amide bond- and peptide bond-cleaving enzymes

    PubMed Central

    Kobayashi, Michihiko; Fujiwara, Yoshie; Goda, Masahiko; Komeda, Hidenobu; Shimizu, Sakayu

    1997-01-01

    Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 → Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 → Asn substitution as well as Ser195 → Ala substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH2) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two. PMID:9342349

  18. Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

    PubMed

    Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

    2003-05-13

    Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate

  19. Monocopper active site for partial methane oxidation in Cu-exchanged 8MR zeolites

    SciTech Connect

    Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; Nørskov, Jens K.; Studt, Felix

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidation reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [CuIIOH]+ species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [CuIIOH]+ active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu]2+ and Cu3O3 motifs.

  20. Monocopper active site for partial methane oxidation in Cu-exchanged 8MR zeolites

    DOE PAGES

    Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; ...

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidationmore » reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [CuIIOH]+ species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [CuIIOH]+ active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu]2+ and Cu3O3 motifs.« less

  1. Endocrine disrupting activities of surface water associated with a West Virginia oil and gas industry wastewater disposal site.

    PubMed

    Kassotis, Christopher D; Iwanowicz, Luke R; Akob, Denise M; Cozzarelli, Isabelle M; Mumford, Adam C; Orem, William H; Nagel, Susan C

    2016-07-01

    Currently, >95% of end disposal of hydraulic fracturing wastewater from unconventional oil and gas operations in the US occurs via injection wells. Key data gaps exist in understanding the potential impact of underground injection on surface water quality and environmental health. The goal of this study was to assess endocrine disrupting activity in surface water at a West Virginia injection well disposal site. Water samples were collected from a background site in the area and upstream, on, and downstream of the disposal facility. Samples were solid-phase extracted, and extracts assessed for agonist and antagonist hormonal activities for five hormone receptors in mammalian and yeast reporter gene assays. Compared to reference water extracts upstream and distal to the disposal well, samples collected adjacent and downstream exhibited considerably higher antagonist activity for the estrogen, androgen, progesterone, glucocorticoid and thyroid hormone receptors. In contrast, low levels of agonist activity were measured in upstream/distal sites, and were inhibited or absent at downstream sites with significant antagonism. Concurrent analyses by partner laboratories (published separately) describe the analytical and geochemical profiling of the water; elevated conductivity as well as high sodium, chloride, strontium, and barium concentrations indicate impacts due to handling of unconventional oil and gas wastewater. Notably, antagonist activities in downstream samples were at equivalent authentic standard concentrations known to disrupt reproduction and/or development in aquatic animals. Given the widespread use of injection wells for end-disposal of hydraulic fracturing wastewater, these data raise concerns for human and animal health nearby.

  2. Endocrine disrupting activities of surface water associated with a West Virginia oil and gas industry wastewater disposal site

    USGS Publications Warehouse

    Kassotis, Christopher D.; Iwanowicz, Luke R.; Akob, Denise M.; Cozzarelli, Isabelle M.; Mumford, Adam; Orem, William H.; Nagel, Susan C.

    2016-01-01

    Currently, >95% of end disposal of hydraulic fracturing wastewater from unconventional oil and gas operations in the US occurs via injection wells. Key data gaps exist in understanding the potential impact of underground injection on surface water quality and environmental health. The goal of this study was to assess endocrine disrupting activity in surface water at a West Virginia injection well disposal site. Water samples were collected from a background site in the area and upstream, on, and downstream of the disposal facility. Samples were solid-phase extracted, and extracts assessed for agonist and antagonist hormonal activities for five hormone receptors in mammalian and yeast reporter gene assays. Compared to reference water extracts upstream and distal to the disposal well, samples collected adjacent and downstream exhibited considerably higher antagonist activity for the estrogen, androgen, progesterone, glucocorticoid and thyroid hormone receptors. In contrast, low levels of agonist activity were measured in upstream/distal sites, and were inhibited or absent at downstream sites with significant antagonism. Concurrent analyses by partner laboratories (published separately) describe the analytical and geochemical profiling of the water; elevated conductivity as well as high sodium, chloride, strontium, and barium concentrations indicate impacts due to handling of unconventional oil and gas wastewater. Notably, antagonist activities in downstream samples were at equivalent authentic standard concentrations known to disrupt reproduction and/or development in aquatic animals. Given the widespread use of injection wells for end-disposal of hydraulic fracturing wastewater, these data raise concerns for human and animal health nearby.

  3. 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.

  4. Young Children's Preference for Television Viewing Compared with Other Activities.

    ERIC Educational Resources Information Center

    Slavenas, Rosemarie

    1987-01-01

    Young children's own perceptions of their preference for television viewing relative to other developmentally appropriate activities were studied through means of a questionnaire. The respondents rated television viewing as a less attractive activity than playing outside, playing with play dough, and building with sand, but few preferred story…

  5. Kinetics of Hydrogen Atom Abstraction from Substrate by an Active Site Thiyl Radical in Ribonucleotide Reductase

    PubMed Central

    2015-01-01

    Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Active E. coli class Ia RNR is an α2β2 complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 → [β-W48] → β-Y356 → α-Y731 → α-Y730 → α-C439) that spans ∼35 Å. To unmask PCET kinetics from rate-limiting conformational changes, we prepared a photochemical RNR containing a [ReI] photooxidant site-specifically incorporated at position 355 ([Re]-β2), adjacent to PCET pathway residue Y356 in β. [Re]-β2 was further modified by replacing Y356 with 2,3,5-trifluorotyrosine to enable photochemical generation and spectroscopic observation of chemically competent tyrosyl radical(s). Using transient absorption spectroscopy, we compare the kinetics of Y· decay in the presence of substrate and wt-α2, Y731F-α2 ,or C439S-α2, as well as with 3′-[2H]-substrate and wt-α2. We find that only in the presence of wt-α2 and the unlabeled substrate do we observe an enhanced rate of radical decay indicative of forward radical propagation. This observation reveals that cleavage of the 3′-C–H bond of substrate by the transiently formed C439· thiyl radical is rate-limiting in forward PCET through α and has allowed calculation of a lower bound for the rate constant associated with this step of (1.4 ± 0.4) × 104 s–1. Prompting radical propagation with light has enabled observation of PCET events heretofore inaccessible, revealing active site chemistry at the heart of RNR catalysis. PMID:25353063

  6. The surface chemistry of heterogeneous catalysis: mechanisms, selectivity, and active sites.

    PubMed

    Zaera, Francisco

    2005-01-01

    The role of chemical kinetics in defining the requirements for the active sites of heterogeneous catalysts is discussed. A personal view is presented, with specific examples from our laboratory to illustrate the role of the chemical composition, structure, and electronic properties of specific surface sites in determining reaction activity and selectivity. Manipulation of catalytic behavior via the addition of chemical modifiers and by tuning of the reaction conditions is also introduced.

  7. 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.

  8. Comparative study of the microbial quality of greywater treated by three on-site treatment systems.

    PubMed

    Friedler, E; Kovalio, R; Ben-Zvi, A

    2006-06-01

    This paper analyses the performance of a pilot scale treatment plant, treating light domestic greywater. The treatment included three parallel treatment units: stand-alone sand filtration (SFEB), RBC followed by sand filtration (SFRBC), and an MBR equipped with UF membranes (MBR). The performance of the SFEB unit was rather poor. The RBC and MBR units produced effluent of excellent quality, with COD of 42 and 40 mg l(-1), BOD of 1.8 and 1.1 mg l(-1), and turbidity of 0.6 and 0.2 NTU respectively. The SFEB failed to remove heterotrophic microorganisms (HPC), while the SFRBC and the MBR exhibited 2.1 and 3.6 logs removal, leading to effluent concentrations of 1.1 x 10(3) and 8.8 x 10(3) cfu ml(-1) respectively. Faecal coliforms (FC) counts were 3.4 x 10(5) 1.4 x 10(5) 1.1 x 10(3) and 3.5 x 10(2) cfu 100 ml(-1) in raw greywater, and in the SFEB, SFRBC and MBR effluents respectively. Further, in 60% of the samples no FC were detected in the MBR effluent. In order to simulate residence times in full scale systems, effluents were disinfected and stored for 0.5 h, 3 h, 6 h (normal operation), and one week (extreme event). The average chlorine demand was 8.1, 3.8 and 2.9 mg l(-1) for SFEB, SFRBC and MBR effluents respectively. Low residual chlorine (0.15-0.22 mg l(-1)) remained in all effluents even after a week-long storage. Disinfection reduced HPC by 5, 2 and 2 orders of magnitude in the SFEB, SFRBC and MBR effluents respectively, with no regrowth in short contact times (up to 6 hours). Some regrowth was observed after a week-long storage leading to 10(6), 10(4) and 10(3) cfu ml(-1) (SFEB SFRBC and MBR respectively). Disinfection reduced FC counts in all three types of effluent to 0 cfu 100 ml(-1), whilst no FC regrowth was observed after week-long storage. The results show that both RBC and MBR treatment units are viable options for on-site greywater reuse. The disinfection experiments strongly indicate that the health risk associated with the reuse of these effluents

  9. Number and locations of agonist binding sites required to activate homomeric Cys-loop receptors.

    PubMed

    Rayes, Diego; De Rosa, María José; Sine, Steven M; Bouzat, Cecilia

    2009-05-06

    Homo-pentameric Cys-loop receptors contain five identical agonist binding sites, each formed at a subunit interface. To determine the number and locations of binding sites required to generate a stable active state, we constructed a receptor subunit with a mutation that disables the agonist binding site and a reporter mutation that alters unitary conductance and coexpressed mutant and nonmutant subunits. Although receptors with a range of different subunit compositions are produced, patch-clamp recordings reveal that the amplitude of each single-channel opening event reports the number and, for certain subunit combinations, the locations of subunits with intact binding sites. We find that receptors with three binding sites at nonconsecutive subunit interfaces exhibit maximal mean channel open time, receptors with binding sites at three consecutive or two nonconsecutive interfaces exhibit intermediate open time, and receptors with binding sites at two consecutive or one interface exhibit brief open time. Macroscopic recordings after rapid application of agonist reveal that channel activation slows and the extent of desensitization decreases as the number of binding sites per receptor decreases. The overall results provide a framework for defining mechanisms of activation and drug modulation for homo-pentameric Cys-loop receptors.

  10. The pH dependence of hairpin ribozyme catalysis reflects ionization of an active site adenine.

    PubMed

    Cottrell, Joseph W; Scott, Lincoln G; Fedor, Martha J

    2011-05-20

    Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pK(a) values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pK(a) for ionization of 8azaA at position 38 with the apparent pK(a) for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK(a) values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5'-oxygen-phosphorus bond.

  11. Direct Visualization of Catalytically Active Sites at the FeO-Pt(111) Interface

    SciTech Connect

    Kudernatsch, Wilhelmine; Peng, Guowen; Zeuthen, Helene; Bai, Yunhai; Merte, L. R.; Lammich, Lutz; Besenbacher, Fleming; Mavrikakis, Manos; Wendt, Stefen

    2015-08-25

    Within the area of surface science, one of the “holy grails” is to directly visualize a chemical reaction at the atomic scale. Whereas this goal has been reached by high-resolution scanning tunneling microscopy (STM) in a number of cases for reactions occurring at flat surfaces, such a direct view is often inhibited for reaction occurring at steps and interfaces. Here we have studied the CO oxidation reaction at the interface between ultrathin FeO islands and a Pt(111) support by in situ STM and density functional theory (DFT) calculations. Time-lapsed STM imaging on this inverse model catalyst in O2 and CO environments revealed catalytic activity occurring at the FeO-Pt(111) interface and directly showed that the Fe-edges host the catalytically most active sites for the CO oxidation reaction. This is an important result since previous evidence for the catalytic activity of the FeO-Pt(111) interface is essentially based on averaging techniques in conjunction with DFT calculations. The presented STM results are in accord with DFTþU calculations, in which we compare possible CO oxidation pathways on oxidized Fe-edges and O-edges. We found that the CO oxidation reaction is more favorable on the oxidized Fe-edges, both thermodynamically and kinetically.

  12. Comparative topical anti-inflammatory activity of cannabinoids and cannabivarins.

    PubMed

    Tubaro, Aurelia; Giangaspero, Anna; Sosa, Silvio; Negri, Roberto; Grassi, Gianpaolo; Casano, Salvatore; Della Loggia, Roberto; Appendino, Giovanni

    2010-10-01

    A selection of seven phytocannabinoids representative of the major structural types of classic cannabinoids and their corresponding cannabivarins was investigated for in vivo topical anti-inflammatory activity in the Croton oil mouse ear dermatitis assay. Differences in the terpenoid moiety were far more important for anti-inflammatory activity than those at the C-3 alkyl residue, suggesting the involvement not only of cannabinoid receptors, but also of other inflammatory end-points targeted by phytocannabinoids.

  13. Comparing bioenergy production sites in the Southeastern US regarding ecosystem service supply and demand.

    PubMed

    Meyer, Markus A; Chand, Tanzila; Priess, Joerg A

    2015-01-01

    Biomass for bioenergy is debated for its potential synergies or tradeoffs with other provisioning and regulating ecosystem services (ESS). This biomass may originate from different production systems and may be purposefully grown or obtained from residues. Increased concerns globally about the sustainable production of biomass for bioenergy has resulted in numerous certification schemes focusing on best management practices, mostly operating at the plot/field scale. In this study, we compare the ESS of two watersheds in the southeastern US. We show the ESS tradeoffs and synergies of plantation forestry, i.e., pine poles, and agricultural production, i.e., wheat straw and corn stover, with the counterfactual natural or semi-natural forest in both watersheds. The plantation forestry showed less distinct tradeoffs than did corn and wheat production, i.e., for carbon storage, P and sediment retention, groundwater recharge, and biodiversity. Using indicators of landscape composition and configuration, we showed that landscape planning can affect the overall ESS supply and can partly determine if locally set environmental thresholds are being met. Indicators on landscape composition, configuration and naturalness explained more than 30% of the variation in ESS supply. Landscape elements such as largely connected forest patches or more complex agricultural patches, e.g., mosaics with shrub and grassland patches, may enhance ESS supply in both of the bioenergy production systems. If tradeoffs between biomass production and other ESS are not addressed by landscape planning, it may be reasonable to include rules in certification schemes that require, e.g., the connectivity of natural or semi-natural forest patches in plantation forestry or semi-natural landscape elements in agricultural production systems. Integrating indicators on landscape configuration and composition into certification schemes is particularly relevant considering that certification schemes are governance

  14. Comparing Bioenergy Production Sites in the Southeastern US Regarding Ecosystem Service Supply and Demand

    PubMed Central

    Meyer, Markus A.; Chand, Tanzila; Priess, Joerg A.

    2015-01-01

    Biomass for bioenergy is debated for its potential synergies or tradeoffs with other provisioning and regulating ecosystem services (ESS). This biomass may originate from different production systems and may be purposefully grown or obtained from residues. Increased concerns globally about the sustainable production of biomass for bioenergy has resulted in numerous certification schemes focusing on best management practices, mostly operating at the plot/field scale. In this study, we compare the ESS of two watersheds in the southeastern US. We show the ESS tradeoffs and synergies of plantation forestry, i.e., pine poles, and agricultural production, i.e., wheat straw and corn stover, with the counterfactual natural or semi-natural forest in both watersheds. The plantation forestry showed less distinct tradeoffs than did corn and wheat production, i.e., for carbon storage, P and sediment retention, groundwater recharge, and biodiversity. Using indicators of landscape composition and configuration, we showed that landscape planning can affect the overall ESS supply and can partly determine if locally set environmental thresholds are being met. Indicators on landscape composition, configuration and naturalness explained more than 30% of the variation in ESS supply. Landscape elements such as largely connected forest patches or more complex agricultural patches, e.g., mosaics with shrub and grassland patches, may enhance ESS supply in both of the bioenergy production systems. If tradeoffs between biomass production and other ESS are not addressed by landscape planning, it may be reasonable to include rules in certification schemes that require, e.g., the connectivity of natural or semi-natural forest patches in plantation forestry or semi-natural landscape elements in agricultural production systems. Integrating indicators on landscape configuration and composition into certification schemes is particularly relevant considering that certification schemes are

  15. Comparing rapid-screening and standard toxicity assays to assess known chemical contamination at a hazardous waste site

    SciTech Connect

    Martino, L.; Swigert, J.; Roberts, C.

    1995-12-31

    The thrust to streamline the Superfund site investigation/remediation program makes it critical for site investigators to utilize rapid screening methodologies to facilitate decision-making. However, screening methodologies providing information upon which decision-making is based must not only be rapid but also scientifically valid. This presentation compares and contrasts two rapid screening toxicity assessments, the Daphnia magna IQ Toxicity Test {trademark} and Microtox{trademark}, to a battery of standard aquatic toxicity tests using Lemna, Rana, Pimephales, Selenastruni and Ceriodaphnia. Chemical analysis of test water samples provided evidence of potential toxicological risk associated with the test samples. The study site was J-Field, Aberdeen Proving Ground, Maryland, a federal facility listed on the National Priority List that used to test and/or dispose of high explosives and chemical warfare agents in open pits or fields. Surface water samples from 20 sites were collected and used in the toxicity assessments. Water samples also were analyzed for explosives, chemical surety degradation compounds, Target Analyte List (inorganics), Target Compound List (organics) and selected pesticides and PCBs. The Microtox{trademark} assay did not reveal any toxicity present in the samples analyzed. Correlation analyses showed only slight correlation between the Daphnia magna IQ{trademark} assay and the standard 48-hour toxicity test. No correlation existed between the Microtox{trademark} assay and the aquatic toxicity tests. Results are discussed in light of the expected risk of the chemicals known to be present and the outcome of the toxicity tests.

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

    PubMed Central

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

    2015-01-01

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

  17. 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

  18. 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.

  19. Comparative Analysis of Protein Tyrosine Phosphatases Regulating Microglial Activation

    PubMed Central

    Song, Gyun Jee; Kim, Jaehong; Kim, Jong-Heon; Song, Seungeun; Park, Hana; Zhang, Zhong-Yin

    2016-01-01

    Protein tyrosine phosphatases (PTPs) are key regulatory factors in inflammatory signaling pathways. Although PTPs have been extensively studied, little is known about their role in neuroinflammation. In the present study, we examined the expression of 6 different PTPs (PTP1B, TC-PTP, SHP2, MEG2, LYP, and RPTPβ) and their role in glial activation and neuroinflammation. All PTPs were expressed in brain and glia. The expression of PTP1B, SHP2, and LYP was enhanced in the inflamed brain. The expression of PTP1B, TC-PTP, and LYP was increased after treating microglia cells with lipopolysaccharide (LPS). To examine the role of PTPs in microglial activation and neuroinflammation, we used specific pharmacological inhibitors of PTPs. Inhibition of PTP1B, TC-PTP, SHP2, LYP, and RPTPβ suppressed nitric oxide production in LPS-treated microglial cells in a dose-dependent manner. Furthermore, intracerebroventricular injection of PTP1B, TC-PTP, SHP2, and RPTPβ inhibitors downregulated microglial activation in an LPS-induced neuroinflammation model. Our results indicate that multiple PTPs are involved in regulating microglial activation and neuroinflammation, with different expression patterns and specific functions. Thus, PTP inhibitors can be exploited for therapeutic modulation of microglial activation in neuroinflammatory diseases. PMID:27790059

  20. Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase – a template for drug design

    PubMed Central

    Saravanamuthu, Ahilan; Vickers, Tim J.; Bond, Charles S.; Peterson, Mark R.; Hunter, William N.; Fairlamb, Alan H.

    2012-01-01

    SUMMARY Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Since this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidised form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 Å, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through π-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with non-planar molecules, such as clomipramine, where stacking is not possible. PMID:15102853

  1. The three Mycobacterium tuberculosis antigen 85 isoforms have unique substrates and activities determined by non-active site regions.

    PubMed

    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

    2014-09-05

    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 Pro(216)-Phe(228) 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.

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

    SciTech Connect

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

    2007-01-01

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

  3. Dopamine transporter comparative molecular modeling and binding site prediction using the LeuT(Aa) leucine transporter as a template.

    PubMed

    Indarte, Martín; Madura, Jeffry D; Surratt, Christopher K

    2008-02-15

    Pharmacological and behavioral studies indicate that binding of cocaine and the amphetamines by the dopamine transporter (DAT) protein is principally responsible for initiating the euphoria and addiction associated with these drugs. The lack of an X-ray crystal structure for the DAT or any other member of the neurotransmitter:sodium symporter (NSS) family has hindered understanding of psychostimulant recognition at the atomic level; structural information has been obtained largely from mutagenesis and biophysical studies. The recent publication of a crystal structure for the bacterial leucine transporter LeuT(Aa), a distantly related NSS family homolog, provides for the first time a template for three-dimensional comparative modeling of NSS proteins. A novel computational modeling approach using the capabilities of the Molecular Operating Environment program MOE 2005.06 in conjunction with other comparative modeling servers generated the LeuT(Aa)-directed DAT model. Probable dopamine and amphetamine binding sites were identified within the DAT model using multiple docking approaches. Binding sites for the substrate ligands (dopamine and amphetamine) overlapped substantially with the analogous region of the LeuT(Aa) crystal structure for the substrate leucine. The docking predictions implicated DAT side chains known to be critical for high affinity ligand binding and suggest novel mutagenesis targets in elucidating discrete substrate and inhibitor binding sites. The DAT model may guide DAT ligand QSAR studies, and rational design of novel DAT-binding therapeutics.

  4. Comparative study between two different active flutter suppression systems

    NASA Technical Reports Server (NTRS)

    Nissim, E.

    1978-01-01

    An activated leading-edge (LE)-tailing-edge (TE) control system is applied to a drone aircraft with the objective of enabling the drone to fly subsonically at dynamic pressures which are 44% above the open-loop flutter dynamic pressure. The control synthesis approach is based on the aerodynamic energy concept and it incorporates recent developments in this area. A comparison is made between the performance of the activated LE-TE control system and the performance of a TE control system, analyzed in a previous work. The results obtained indicate that although all the control systems achieve the flutter suppression objectives, the TE control system appears to be somewhat superior to the LE-TE control system, in this specific application. This superiority is manifested through reduced values of control surface activity over a wide range of flight conditions.

  5. 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.

  6. All the catalytic active sites of MoS2 for hydrogen evolution

    SciTech Connect

    Li, Guoqing; Zhang, Du; Qiao, Qiao; Yu, Yifei; Peterson, David; Zafar, Abdullah; Kumar, Raj; Curtarolo, Stefano; Hunte, Frank; Shannon, Steve; Zhu, Yimei; Yang, Weitao; Cao, Linyou

    2016-11-29

    MoS2 presents a promising low-cost catalyst for the hydrogen evolution reaction (HER), but the understanding about its active sites has remained limited. Here we present an unambiguous study of the catalytic activities of all possible reaction sites of MoS2, including edge sites, sulfur vacancies, and grain boundaries. We demonstrate that, in addition to the well-known catalytically active edge sites, sulfur vacancies provide another major active site for the HER, while the catalytic activity of grain boundaries is much weaker. Here, the intrinsic turnover frequencies (Tafel slopes) of the edge sites, sulfur vacancies, and grain boundaries are estimated to be 7.5 s–1 (65–75 mV/dec), 3.2 s–1 (65–85 mV/dec), and 0.1 s–1 (120–160 mV/dec), respectively. We also demonstrate that the catalytic activity of sulfur vacancies strongly depends on the density of the vacancies and the local crystalline structure in proximity to the vacancies. Unlike edge sites, whose catalytic activity linearly depends on the length, sulfur vacancies show optimal catalytic activities when the vacancy density is in the range of 7–10%, and the number of sulfur vacancies in high crystalline quality MoS2 is higher than that in low crystalline quality MoS2, which may be related with the proximity of different local crystalline structures to the vacancies.

  7. Active VSG expression sites in Trypanosoma brucei are depleted of nucleosomes.

    PubMed

    Stanne, Tara M; Rudenko, Gloria

    2010-01-01

    African trypanosomes regulate transcription differently from other eukaryotes. Most of the trypanosome genome is constitutively transcribed by RNA polymerase II (Pol II) as large polycistronic transcription units while the genes encoding the major surface proteins are transcribed by RNA polymerase I (Pol I). In bloodstream form Trypanosoma brucei, the gene encoding the variant surface glycoprotein (VSG) coat is expressed in a monoallelic fashion from one of about 15 VSG bloodstream form expression sites (BESs). Little is known about the chromatin structure of the trypanosome genome, and the chromatin state of active versus silent VSG BESs remains controversial. Here, we determined histone H3 occupancy within the genome of T. brucei, focusing on active versus silent VSG BESs in the bloodstream form. We found that histone H3 was most enriched in the nontranscribed 50-bp and 177-bp repeats and relatively depleted in Pol I, II, and III transcription units, with particular depletion over promoter regions. Using two isogenic T. brucei lines containing marker genes in different VSG BESs, we determined that histone H3 is 11- to 40-fold depleted from active VSG BESs compared with silent VSG BESs. Quantitative PCR analysis of fractionated micrococcal nuclease-digested chromatin revealed that the active VSG BES is depleted of nucleosomes. Therefore, in contrast to earlier views, nucleosome positioning appears to be involved in the monoalleleic control of VSG BESs in T. brucei. This may provide a level of epigenetic regulation enabling bloodstream form trypanosomes to efficiently pass on the transcriptional state of active and silent BESs to daughter cells.

  8. A Comparative Study of Active Play on Differently Designed Playgrounds

    ERIC Educational Resources Information Center

    Luchs, Antje; Fikus, Monika

    2013-01-01

    The physical and social environment of children in cities is continuously changing. Knowledge about the positive effects of natural play experiences within the child's development is becoming widely known. Affordances of diverse landscape elements and especially loose parts for play in natural environments influence play activities. New concepts…

  9. Comparing Team Learning Approaches through the Lens of Activity Theory

    ERIC Educational Resources Information Center

    Park, Sunyoung; Cho, Yonjoo; Yoon, Seung Won; Han, Heeyoung

    2013-01-01

    Purpose: The purpose of this study is to examine the distinctive features of three team learning approaches (action learning, problem-based learning, and project-based learning), compare and contrast them, and discuss implications for practice and research. Design/methodology/approach: The authors used Torraco's integrative literature review…

  10. Comparing nocturnal eddy covariance measurements to estimates of ecosystem respiration made by scaling chamber measurements at six coniferous boreal sites

    USGS Publications Warehouse

    Lavigne, M.B.; Ryan, M.G.; Anderson, D.E.; Baldocchi, D.D.; Crill, P.M.; Fitzjarrald, D.R.; Goulden, M.L.; Gower, S.T.; Massheder, J.M.; McCaughey, J.H.; Rayment, M.; Striegl, R. G.

    1997-01-01

    During the growing season, nighttime ecosystem respiration emits 30–100% of the daytime net photosynthetic uptake of carbon, and therefore measurements of rates and understanding of its control by the environment are important for understanding net ecosystem exchange. Ecosystem respiration can be measured at night by eddy covariance methods, but the data may not be reliable because of low turbulence or other methodological problems. We used relationships between woody tissue, foliage, and soil respiration rates and temperature, with temperature records collected on site to estimate ecosystem respiration rates at six coniferous BOREAS sites at half-hour or 1-hour intervals, and then compared these estimates to nocturnal measurements of CO2 exchange by eddy covariance. Soil surface respiration was the largest source of CO2 at all sites (48–71%), and foliar respiration made a large contribution to ecosystem respiration at all sites (25–43%). Woody tissue respiration contributed only 5–15% to ecosystem respiration. We estimated error for the scaled chamber predictions of ecosystem respiration by using the uncertainty associated with each respiration parameter and respiring biomass value. There was substantial uncertainty in estimates of foliar and soil respiration because of the spatial variability of specific respiration rates. In addition, more attention needs to be paid to estimating foliar respiration during the early part of the growing season, when new foliage is growing, and to determining seasonal trends of soil surface respiration. Nocturnal eddy covariance measurements were poorly correlated to scaled chamber estimates of ecosystem respiration (r2=0.06–0.27) and were consistently lower than scaled chamber predictions (by 27% on average for the six sites). The bias in eddy covariance estimates of ecosystem respiration will alter estimates of gross assimilation in the light and of net ecosystem exchange rates over extended periods.

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

    SciTech Connect

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

    2010-04-30

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

  12. Quantum mechanics study of the hydroxyethylamines-BACE-1 active site interaction energies.

    PubMed

    Gueto-Tettay, Carlos; Drosos, Juan Carlos; Vivas-Reyes, Ricardo

    2011-06-01

    The identification of BACE-1, a key enzyme in the production of Amyloid-β (Aβ) peptides, generated by the proteolytic processing of amyloid precursor protein, was a major advance in the field of Alzheimer's disease as this pathology is characterized by the presence of extracellular senile plaques, mainly comprised of Aβ peptides. Hydroxyethylamines have demonstrated a remarkable potential, like candidate drugs, for this disease using BACE-1 as target. Density Functional Theory calculations were employed to estimate interaction energies for the complexes formed between the hydroxyethylamine derivated inhibitors and 24 residues in the BACE-1 active site. The collected data offered not only a general but a particular quantitative description that gives a deep insight of the interactions in the active site, showing at the same time how ligand structural variations affect them. Polar interactions are the major energetic contributors for complex stabilization and those ones with charged aspartate residues are highlighted, as they contribute over 90% of the total attractive interaction energy. Ligand-ARG296 residue interaction reports the most repulsive value and decreasing of the magnitude of this repulsion can be a key feature for the design of novel and more potent BACE-1 inhibitors. Also it was explained why sultam derivated BACE-1 inhibitors are better ones than lactam based. Hydrophobic interactions concentrated at S1 zone and other relevant repulsions and attractions were also evaluated. The comparison of two different theory levels (X3LYP and M062X) allowed to confirm the relevance of the detected interactions as each theory level has its own strength to depict the forces involved, as is the case of M062X which is better describing the hydrophobic interactions. Those facts were also evaluated and confirmed by comparing the quantitative trend, of selected ligand-residue interactions, with MP2 theory level as reference standard method for electrostatic plus

  13. Quantum mechanics study of the hydroxyethylamines-BACE-1 active site interaction energies

    NASA Astrophysics Data System (ADS)

    Gueto-Tettay, Carlos; Drosos, Juan Carlos; Vivas-Reyes, Ricardo

    2011-06-01

    The identification of BACE-1, a key enzyme in the production of Amyloid-β (Aβ) peptides, generated by the proteolytic processing of amyloid precursor protein, was a major advance in the field of Alzheimer's disease as this pathology is characterized by the presence of extracellular senile plaques, mainly comprised of Aβ peptides. Hydroxyethylamines have demonstrated a remarkable potential, like candidate drugs, for this disease using BACE-1 as target. Density Functional Theory calculations were employed to estimate interaction energies for the complexes formed between the hydroxyethylamine derivated inhibitors and 24 residues in the BACE-1 active site. The collected data offered not only a general but a particular quantitative description that gives a deep insight of the interactions in the active site, showing at the same time how ligand structural variations affect them. Polar interactions are the major energetic contributors for complex stabilization and those ones with charged aspartate residues are highlighted, as they contribute over 90% of the total attractive interaction energy. Ligand-ARG296 residue interaction reports the most repulsive value and decreasing of the magnitude of this repulsion can be a key feature for the design of novel and more potent BACE-1 inhibitors. Also it was explained why sultam derivated BACE-1 inhibitors are better ones than lactam based. Hydrophobic interactions concentrated at S1 zone and other relevant repulsions and attractions were also evaluated. The comparison of two different theory levels (X3LYP and M062X) allowed to confirm the relevance of the detected interactions as each theory level has its own strength to depict the forces involved, as is the case of M062X which is better describing the hydrophobic interactions. Those facts were also evaluated and confirmed by comparing the quantitative trend, of selected ligand-residue interactions, with MP2 theory level as reference standard method for electrostatic plus

  14. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  15. Elucidating the mechanism and active site of the cyclohexanol dehydrogenation on copper-based catalysts: A density functional theory study

    NASA Astrophysics Data System (ADS)

    Wang, Ziyun; Liu, Xinyi; Rooney, D. W.; Hu, P.

    2015-10-01

    The dehydrogenation of cyclohexanol to cyclohexanone is very important in the manufacture of nylon. Copper-based catalysts are the most popular catalysts for this reaction, and on these catalysts the reaction mechanism and active site are in debate. In order to elucidate the mechanism and active site of the cyclohexanol dehydrogenation on copper-based catalysts, density functional theory with dispersion corrections were performed on up to six facets of copper in two different oxidation states: monovalent copper and metallic copper. By calculating the surface energies of these facets, Cu(111) and Cu2O(111) were found to be the most stable facets for metallic copper and for monovalent copper, respectively. On these two facets, all the possible elementary steps in the dehydrogenation pathway of cyclohexanol were calculated, including the adsorption, dehydrogenation, hydrogen coupling and desorption. Two different reaction pathways for dehydrogenation were considered on both surfaces. It was revealed that the dehydrogenation mechanisms are different on these two surfaces: on Cu(111) the hydrogen belonging to the hydroxyl is removed first, then the hydrogen belonging to the carbon is subtracted, while on Cu2O(111) the hydrogen belonging to the carbon is removed followed by the subtraction of the hydrogen in the hydroxyl group. Furthermore, by comparing the energy profiles of these two surfaces, Cu2O(111) was found to be more active for cyclohexanol dehydrogenation than Cu(111). In addition, we found that the coordinatively unsaturated copper sites on Cu2O(111) are the reaction sites for all the steps. Therefore, the coordinatively unsaturated copper site on Cu2O(111) is likely to be the active site for cyclohexanol dehydrogenation on the copper-based catalysts.

  16. Identification of a Bidirectional Splicing Enhancer: Differential Involvement of SR Proteins in 5′ or 3′ Splice Site Activation

    PubMed Central

    Bourgeois, Cyril F.; Popielarz, Michel; Hildwein, Georges; Stevenin, James

    1999-01-01

    The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5′ splice sites and of one major or one minor 3′ splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5′ splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5′ splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3′ splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5′ splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity. PMID:10523623

  17. Comparing the influence of sunspot activity and geomagnetic activity on winter surface climate

    NASA Astrophysics Data System (ADS)

    Roy, I.; Asikainen, T.; Maliniemi, V.; Mursula, K.

    2016-11-01

    We compare here the effect of geomagnetic activity (using the aa index) and sunspot activity on surface climate using sea level pressure dataset from Hadley centre during northern winter. Previous studies using the multiple linear regression method have been limited to using sunspots as a solar activity predictor. Sunspots and total solar irradiance indicate a robust positive influence around the Aleutian Low. This is valid up to a lag of one year. However, geomagnetic activity yields a positive NAM pattern at high to polar latitudes and a positive signal around Azores High pressure region. Interestingly, while there is a positive signal around Azores High for a 2-year lag in sunspots, the strongest signal in this region is found for aa index at 1-year lag. There is also a weak but significant negative signature present around central Pacific for both sunspots and aa index. The combined influence of geomagnetic activity and Quasi Biannual Oscillation (QBO 30 hPa) produces a particularly strong response at mid to polar latitudes, much stronger than the combined influence of sunspots and QBO, which was mostly studied in previous studies so far. This signal is robust and insensitive to the selected time period during the last century. Our results provide a useful way for improving the prediction of winter weather at middle to high latitudes of the northern hemisphere.

  18. Comparing Educational Tools Using Activity Theory: Clickers and Flashcards

    NASA Astrophysics Data System (ADS)

    Price, Edward; De Leone, Charles; Lasry, Nathaniel

    2010-10-01

    Physics educators and researchers have recently begun to distinguish between pedagogical approaches and the educational technologies that are used to implement them. For instance, peer instruction has been shown to be equally effective, in terms of student learning outcomes, when implemented with clickers or flashcards. Therefore, technological tools (clickers and flashcards) can be viewed as means to mediate pedagogical techniques (peer instruction or traditional instruction). In this paper, we use activity theory to examine peer instruction, with particular attention to the role of tools. This perspective helps clarify clickers' and flashcards' differences, similarities, impacts in the classroom, and utility to education researchers. Our analysis can suggest improvements and new uses. Finally, we propose activity theory as a useful approach in understanding and improving the use of technology in the physics classroom.

  19. Plantar pressures during level walking compared with other ambulatory activities.

    PubMed

    Lundeen, S; Lundquist, K; Cornwall, M W; McPoil, T G

    1994-06-01

    This study was designed to determine the magnitude of plantar pressures during level walking in comparison to other activities. These activities included climbing up stairs, going down stairs, a simple pivot while walking, and a crossover pivot while walking in normal individuals. Twelve volunteers, six men and six women, mean age 28 years, served as subjects. Data were collected on the dominant foot with an EMED-SF pressure sensor platform as each subject walked barefoot and did each of the five activities. Maximum plantar pressure (MPP) and pressure-time integral (PTI) was found in the metatarsal and heel regions. The results of repeated-measures analysis of variance tests showed that the five experimental conditions were statistically different for both MPP and PTI in the metatarsal and heel regions. Post hoc analysis indicated that MPP and PTI were decreased during the going down stairs condition in the heel and increased during the crossover pivot while walking and pivot while walking conditions for the metatarsal region.

  20. 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.

  1. Synergistic effect between defect sites and functional groups on the hydrolysis of cellulose over activated carbon.

    PubMed

    Foo, Guo Shiou; Sievers, Carsten

    2015-02-01

    The chemical oxidation of activated carbon by H2 O2 and H2 SO4 is investigated, structural and chemical modifications are characterized, and the materials are used as catalysts for the hydrolysis of cellulose. Treatment with H2 O2 enlarges the pore size and imparts functional groups such as phenols, lactones, and carboxylic acids. H2 SO4 treatment targets the edges of carbon sheets primarily, and this effect is more pronounced with a higher temperature. Adsorption isotherms demonstrate that the adsorption of oligomers on functionalized carbon is dominated by van der Waals forces. The materials treated chemically are active for the hydrolysis of cellulose despite the relative weakness of most of their acid sites. It is proposed that a synergistic effect between defect sites and functional groups enhances the activity by inducing a conformational change in the glucan chains if they are adsorbed at defect sites. This activates the glycosidic bonds for hydrolysis by in-plane functional groups.

  2. 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

  3. Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site

    PubMed Central

    Ma, Yi; Zhang, Beilei; Wang, Meng; Ou, Yanghui

    2016-01-01

    The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) with 5′-3′ DNA polymerase activity while in absence of 5′-3′ exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified in E. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas. PMID:27981047

  4. Comparative efficacy of switching to natalizumab in active multiple sclerosis

    PubMed Central

    Spelman, Timothy; Kalincik, Tomas; Zhang, Annie; Pellegrini, Fabio; Wiendl, Heinz; Kappos, Ludwig; Tsvetkova, Larisa; Belachew, Shibeshih; Hyde, Robert; Verheul, Freek; Grand-Maison, Francois; Izquierdo, Guillermo; Grammond, Pierre; Duquette, Pierre; Lugaresi, Alessandra; Lechner-Scott, Jeannette; Oreja-Guevara, Celia; Hupperts, Raymond; Petersen, Thor; Barnett, Michael; Trojano, Maria; Butzkueven, Helmut

    2015-01-01

    Objective To compare treatment efficacy and persistence in patients who switched to natalizumab versus those who switched between glatiramer acetate (GA) and interferon-beta (IFNβ) after an on-treatment relapse on IFNβ or GA using propensity score matched real-world datasets. Methods Patients included were registered in MSBase or the TYSABRI Observational Program (TOP), had relapsed on IFNβ or GA within 12 months prior to switching to another therapy, and had initiated natalizumab or IFNβ/GA treatment ≤6 months after discontinuing prior therapy. Covariates were balanced across post switch treatment groups by propensity score matching at treatment initiation. Relapse, persistence, and disability measures were compared between matched treatment arms in the total population (n = 869/group) and in subgroups defined by prior treatment history (IFNβ only [n = 578/group], GA only [n = 165/group], or both IFNβ and GA [n = 176/group]). Results Compared to switching between IFNβ and GA, switching to natalizumab reduced annualized relapse rate in year one by 65–75%, the risk of first relapse by 53–82% (mean follow-up 1.7–2.2 years) and treatment discontinuation events by 48–65% (all P ≤ 0.001). In the total population, switching to natalizumab reduced the risk of confirmed disability progression by 26% (P = 0.036) and decreased the total disability burden by 1.54 EDSS-years (P < 0.0001) over the first 24 months post switch. Interpretation Using large, real-world, propensity-matched datasets we demonstrate that after a relapse on IFNβ or GA, switching to natalizumab (rather than between IFNβ and GA) led to superior outcomes for patients in all measures assessed. Results were consistent regardless of the prior treatment identity. PMID:25909083

  5. The Unexpected Reactivity of the Carbon Sites on the Nanostructured Carbon Catalysts towards the C-H Bond Activation from the Analysis of the Aromaticity.

    PubMed

    Sun, XiaoYing; Li, Bo; Su, DangSheng

    2016-06-06

    It is believed that the oxygen groups on the carbon catalysts are responsible for the observed reactivity for C-H bond activations. On the other hand, the oxygen groups also reduce the aromaticity of the host. The loss of the aromaticity increases reactivities of the carbon atoms and they become the active sites for the C-H bond activation. The newly identified C-C site exhibits the comparable catalytic performance in the oxidative dehydrogenation (ODH) of propane compared with the conventional oxygen groups like quinone and ketone. A series of calculations indicate that the aromaticity might be a useful descriptor for the carbon catalysts.

  6. Effects of sex and site on amino acid metabolism enzyme gene expression and activity in rat white adipose tissue

    PubMed Central

    Arriarán, Sofía; Agnelli, Silvia; Remesar, Xavier; Fernández-López, José Antonio

    2015-01-01

    Background and Objectives. White adipose tissue (WAT) shows marked sex- and diet-dependent differences. However, our metabolic knowledge of WAT, especially on amino acid metabolism, is considerably limited. In the present study, we compared the influence of sex on the amino acid metabolism profile of the four main WAT sites, focused on the paths related to ammonium handling and the urea cycle, as a way to estimate the extent of WAT implication on body amino-nitrogen metabolism. Experimental Design. Adult female and male rats were maintained, undisturbed, under standard conditions for one month. After killing them under isoflurane anesthesia. WAT sites were dissected and weighed. Subcutaneous, perigonadal, retroperitoneal and mesenteric WAT were analyzed for amino acid metabolism gene expression and enzyme activities. Results. There was a considerable stability of the urea cycle activities and expressions, irrespective of sex, and with only limited influence of site. Urea cycle was more resilient to change than other site-specialized metabolic pathways. The control of WAT urea cycle was probably related to the provision of arginine/citrulline, as deduced from the enzyme activity profiles. These data support a generalized role of WAT in overall amino-N handling. In contrast, sex markedly affected WAT ammonium-centered amino acid metabolism in a site-related way, with relatively higher emphasis in males’ subcutaneous WAT. Conclusions. We found that WAT has an active amino acid metabolism. Its gene expressions were lower than those of glucose-lipid interactions, but the differences were quantitatively less important than usually reported. The effects of sex on urea cycle enzymes expression and activity were limited, in contrast with the wider variations observed in other metabolic pathways. The results agree with a centralized control of urea cycle operation affecting the adipose organ as a whole. PMID:26587356

  7. A comparative Study of Circulation Patterns at Active Lava Lakes

    NASA Astrophysics Data System (ADS)

    Lev, Einat; Oppenheimer, Clive; Spampinato, Letizia; Hernandez, Pedro; Unglert, Kathi

    2016-04-01

    Lava lakes present a rare opportunity to study magma dynamics in a large scaled-up "crucible" and provide a unique natural laboratory to ground-truth dynamic models of magma circulation. The persistence of lava lakes allows for long-term observations of flow dynamics and of lava properties, especially compared to surface lava flows. There are currently five persistent lava lakes in the world: Halemaumau in Kilauea (Hawaii, USA), Erta Ale (Ethiopia), Nyiragongo (Congo), Erebus (Antarctica), and Villarica (Chile). Marum and Benbow craters of Ambrym volcano (Vanuatu) and Masaya (Nicaragua) have often hosted lava lakes as well. We use visible-light and thermal infrared time-lapse and video footage collected at all above lakes (except Villarica, where the lake is difficult to observe), and compare the circulation patterns recorded. We calculate lake surface motion from the footage using the optical flow method (Lev et al., 2012) to produce 2D velocity fields. We mined both the surface temperature field and the surface velocity field for patterns using machine learning techniques such as "self-organizing maps (SOMs)" and "principle component analysis (PCA)". We use automatic detection technique to study the configuration of crustal plates at the lakes' surface. We find striking differences among the lakes, in flow direction, flow speed, frequency of changes in flow direction and speed, location and consistency of upwelling and downwelling, and crustal plate configuration. We relate the differences to lake size, shallow conduit geometry, lava viscosity, crystal and gas content, and crust integrity.

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

    PubMed

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

    2012-05-01

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

  9. 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.

  10. 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.

  11. Transcriptional activation by LR1 at the Eµ enhancer and switch region sites

    PubMed Central

    Hanakahi, L. A.; Maizels, Nancy

    2000-01-01

    LR1 is a B cell-specific, sequence-specific duplex DNA binding activity which is induced in B cells carrying out class switch recombination. Here we identify several properties of LR1 which enable it to function in transcriptional regulation. We show that LR1 contributes to transcriptional activation by the Eµ immunoglobulin heavy chain intron enhancer by binding to a site within the enhancer core. We further show that LR1 bends DNA upon binding. In addition, we show that LR1 is itself a bona fide transcriptional activator, as multimerized LR1 sites produce an element which can enhance transcription from a minimal promoter. In order for class switch recombination to occur, an activating signal must be transmitted via the Eµ core, and both S regions targeted for recombination must be actively transcribed. The properties of LR1 that we have identified suggest distinct potential functions of LR1 duplex DNA binding activity in class switch recombination. First, LR1 may contribute to recombinational activation by the Eµ core. Second, there are multiple potential LR1 duplex binding sites in each of the G-rich switch regions, and LR1 bound at contiguous sites may enhance recombination by stimulating transcription of the S regions. PMID:10908319

  12. Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

    PubMed Central

    Sopory, Shailaja; Kwon, Sunjong; Wehrli, Marcel; Christian, Jan L.

    2010-01-01

    BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 site of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range. PMID:20659445

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

    Mlýnský, Vojtěch; Šponer, Jiří

    2014-01-01

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

  15. Phosphorylation of serine 264 impedes active site accessibility in the E1 component of the human pyruvate dehydrogenase multienzyme complex.

    PubMed

    Seifert, Franziska; Ciszak, Ewa; Korotchkina, Lioubov; Golbik, Ralph; Spinka, Michael; Dominiak, Paulina; Sidhu, Sukhdeep; Brauer, Johanna; Patel, Mulchand S; Tittmann, Kai

    2007-05-29

    At the junction of glycolysis and the Krebs cycle in cellular metabolism, the pyruvate dehydrogenase multienzyme complex (PDHc) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. In mammals, PDHc is tightly regulated by phosphorylation-dephosphorylation of three serine residues in the thiamin-dependent pyruvate dehydrogenase (E1) component. In vivo, inactivation of human PDHc correlates mostly with phosphorylation of serine 264, which is located at the entrance of the substrate channel leading to the active site of E1. Despite intense investigations, the molecular mechanism of this inactivation has remained enigmatic. Here, a detailed analysis of microscopic steps of catalysis in human wild-type PDHc-E1 and pseudophosphorylation variant Ser264Glu elucidates how phosphorylation of Ser264 affects catalysis. Whereas the intrinsic reactivity of the active site in catalysis of pyruvate decarboxylation remains nearly unaltered, the preceding binding of substrate to the enzyme's active site via the substrate channel and the subsequent reductive acetylation of the E2 component are severely slowed in the phosphorylation variant. The structure of pseudophosphorylation variant Ser264Glu determined by X-ray crystallography reveals no differences in the three-dimensional architecture of the phosphorylation loop or of the active site, when compared to those of the wild-type enzyme. However, the channel leading to the active site is partially obstructed by the side chain of residue 264 in the variant. By analogy, a similar obstruction of the substrate channel can be anticipated to result from a phosphorylation of Ser264. The kinetic and thermodynamic results in conjunction with the structure of Ser264Glu suggest that phosphorylation blocks access to the active site by imposing a steric and electrostatic barrier for substrate binding and active site coupling with the E2 component. As a Ser264Gln variant, which carries no charge at position 264, is also selectively

  16. An Electromagnetic Interference Study of Potential Transmitter Sites for the HF Active Auroral Research Program (HAARP)

    DTIC Science & Technology

    1993-07-19

    heating . The measurements described in this report were conducted at a number of candidate HAARP transmitter sites in the vicinity of Fairbanks...employ the High Power Auroral Stimulation (HIPAS) RF heating facility [1], located in the Chena River valley area near Fairbanks. HAARP will be an...Potential Transmitter Sites for the HF Active Auroral Research Program ( HAARP ) JOSEP11 A. GOLDSTEIN EDWARD 1. KENNEDY ADRIAN S. ELEY 4 IMICHlAEL A. RuPAR C

  17. Comparative activity profiles of Thielavia terrestris and Trichoderma reesei cellulases

    SciTech Connect

    Tuse, D.; Mason, B.J.; Skinner, W.A.

    1980-10-01

    The successful utilization of cellulosic materials depends on the development of economically feasible processes for the literation of low molecular weight soluble products from the polymers. These soluble products, such as hexoses and pentoses, can then be utilized as substrates for the microbial or chemical product of fuels, food, and chemical feedstocks. In the enzymatic saccharification of cellulose, one of the major roadblocks to the development of commercially attractive processes has been the instability of the cellulase complex. It is desirable, for example, to operate the conversion systems at elevated temperatures, but environments with high thermal energy can significantly shorten enzyme half life. The authors have isolated a strain of the fungus Thielavia terrestris that possesses a complete cellulase system, and its enzymes were found to have remarkable thermal stability. The author presents a comparison of the activities of the T. terrestris enzymes with those of Trichoderma reesei.

  18. Probing the catalytic mechanism of bovine CD38/NAD+ glycohydrolase by site directed mutagenesis of key active site residues.

    PubMed

    Kuhn, Isabelle; Kellenberger, Esther; Cakir-Kiefer, Céline; Muller-Steffner, Hélène; Schuber, Francis

    2014-07-01

    Bovine CD38/NAD(+) glycohydrolase catalyzes the hydrolysis of NAD(+) to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism. Our recent crystallographic study of its Michaelis complex and covalently-trapped intermediates provided insights into the modalities of substrate binding and the molecular mechanism of bCD38. The aim of the present work was to determine the precise role of key conserved active site residues (Trp118, Glu138, Asp147, Trp181 and Glu218) by focusing mainly on the cleavage of the nicotinamide-ribosyl bond. We analyzed the kinetic parameters of mutants of these residues which reside within the bCD38 subdomain in the vicinity of the scissile bond of bound NAD(+). To address the reaction mechanism we also performed chemical rescue experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. The crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2'-OH group of NAD(+), was highlighted. This contribution to catalysis accounts for almost half of the reaction energy barrier. Other contributions can be ascribed notably to Glu138 and Asp147 via ground-state destabilization and desolvation in the vicinity of the scissile bond. Key interactions with Trp118 and Trp181 were also proven to stabilize the ribooxocarbenium ion-like transition state. Altogether we propose that, as an alternative to a covalent acylal reaction intermediate with Glu218, catalysis by bCD38 proceeds through the formation of a discrete and transient ribooxocarbenium intermediate which is stabilized within the active site mostly by electrostatic interactions.

  19. 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

  20. Cost Analysis and Surgical Site Infection Rates in Total Knee Arthroplasty Comparing Traditional vs. Single-Use Instrumentation.

    PubMed

    Siegel, Geoffrey W; Patel, Neil N; Milshteyn, Michael A; Buzas, David; Lombardo, Daniel J; Morawa, Lawrence G

    2015-12-01

    Surgical site infections (SSIs) are a significant complications in total knee arthroplasty (TKA). The purpose of this study was to evaluate if traditional vs. single-use instrumentation had an effect on SSI's. We compared SSI rates and costs of TKAs performed with single-use (449) and traditional (169) TKA instrumentation trays. Total OR Time was, on average, 30 min less when single-use instrumentation was used. SSIs decreased in the single-use group (n=1) compared to the traditional group (n=5) (P=0.006). Single-use instrumentation added $490 in initial costs; however it saved between $480 and $600. Single-use instrumentation may provide a benefit to the patient by potentially decreasing the risk of infection and reducing the overall hospital costs.

  1. Time dependent analysis of assay comparability: a novel approach to understand intra- and inter-site variability over time

    NASA Astrophysics Data System (ADS)

    Winiwarter, Susanne; Middleton, Brian; Jones, Barry; Courtney, Paul; Lindmark, Bo; Page, Ken M.; Clark, Alan; Landqvist, Claire

    2015-09-01

    We demonstrate here a novel use of statistical tools to study intra- and inter-site assay variability of five early drug metabolism and pharmacokinetics in vitro assays over time. Firstly, a tool for process control is presented. It shows the overall assay variability but allows also the following of changes due to assay adjustments and can additionally highlight other, potentially unexpected variations. Secondly, we define the minimum discriminatory difference/ratio to support projects to understand how experimental values measured at different sites at a given time can be compared. Such discriminatory values are calculated for 3 month periods and followed over time for each assay. Again assay modifications, especially assay harmonization efforts, can be noted. Both the process control tool and the variability estimates are based on the results of control compounds tested every time an assay is run. Variability estimates for a limited set of project compounds were computed as well and found to be comparable. This analysis reinforces the need to consider assay variability in decision making, compound ranking and in silico modeling.

  2. Comparative aspects of adult neural stem cell activity in vertebrates.

    PubMed

    Grandel, Heiner; Brand, Michael

    2013-03-01

    At birth or after hatching from the egg, vertebrate brains still contain neural stem cells which reside in specialized niches. In some cases, these stem cells are deployed for further postnatal development of parts of the brain until the final structure is reached. In other cases, postnatal neurogenesis continues as constitutive neurogenesis into adulthood leading to a net increase of the number of neurons with age. Yet, in other cases, stem cells fuel neuronal turnover. An example is protracted development of the cerebellar granular layer in mammals and birds, where neurogenesis continues for a few weeks postnatally until the granular layer has reached its definitive size and stem cells are used up. Cerebellar growth also provides an example of continued neurogenesis during adulthood in teleosts. Again, it is the granular layer that grows as neurogenesis continues and no definite adult cerebellar size is reached. Neuronal turnover is most clearly seen in the telencephalon of male canaries, where projection neurons are replaced in nucleus high vocal centre each year before the start of a new mating season--circuitry reconstruction to achieve changes of the song repertoire in these birds? In this review, we describe these and other examples of adult neurogenesis in different vertebrate taxa. We also compare the structure of the stem cell niches to find common themes in their organization despite different functions adult neurogenesis serves in different species. Finally, we report on regeneration of the zebrafish telencephalon after injury to highlight similarities and differences of constitutive neurogenesis and neuronal regeneration.

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

    PubMed

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

    2016-09-01

    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.

  4. 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.

  5. Kinetics of nucleotide entry into RNA polymerase active site provides mechanism for efficiency and fidelity.

    PubMed

    Wang, Beibei; Sexton, Rachel E; Feig, Michael

    2017-04-01

    During transcription, RNA polymerase II elongates RNA by adding nucleotide triphosphates (NTPs) complementary to a DNA template. Structural studies have suggested that NTPs enter and exit the active site via the narrow secondary pore but details have remained unclear. A kinetic model is presented that integrates molecular dynamics simulations with experimental data. Previous simulations of trigger loop dynamics and the dynamics of matched and mismatched NTPs in and near the active site were combined with new simulations describing NTP exit from the active site via the secondary pore. Markov state analysis was applied to identify major states and estimate kinetic rates for transitions between those states. The kinetic model predicts elongation and misincorporation rates in close agreement with experiment and provides mechanistic hypotheses for how NTP entry and exit via the secondary pore is feasible and a key feature for achieving high elongation and low misincorporation rates during RNA elongation.

  6. 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

  7. Gamma exposure rates due to neutron activation of soil: site of Hood detonation, Operation Plumbbob

    SciTech Connect

    Auxier, J.A.; Ohnesorge, W.F.

    1980-06-01

    This paper is the result of some recent discussions of exposure rates within the first few hours of the Hood detonation of the Plumbbob series due to neutron activation of soil. We estimated the exposure rates from 1/2 to 3 h after the detonation from ground zero to 1000 yards from ground zero. The area was assumed to be uncontaminated by fallout. Soil samples from the area of the Nevada Test Site at which the Hood device was detonated were sent to ORNL by Dr. John Malik of Los Alamos and by Mr. Gordon Jacks of the Nevada Test Site. These samples were irradiated at the DOSAR facility and the resulting activity analyzed. Calculations of exposure rates were then made based on the analyzed activity and the measured thermal neutron fluences at DOSAR and at the Hood Site.

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

    PubMed Central

    Petrosino, J F; Palzkill, T

    1996-01-01

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

  9. The effect of the distance between acidic site and basic site immobilized on mesoporous solid on the activity in catalyzing aldol condensation

    SciTech Connect

    Yu Xiaofang; Yu Xiaobo; Wu Shujie; Liu Bo; Liu Heng; Guan Jingqi; Kan Qiubin

    2011-02-15

    Acid-base bifunctional heterogeneous catalysts containing carboxylic and amine groups, which were immobilized at defined distance from one another on the mesoporous solid were synthesized by immobilizing lysine onto carboxyl-SBA-15. The obtained materials were characterized by X-ray diffraction (XRD), N{sub 2} adsorption, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), scanning electron micrographs (SEM), transmission electron micrographs (TEM), elemental analysis, and back titration. Proximal-C-A-SBA-15 with a proximal acid-base distance was more active than maximum-C-A-SBA-15 with a maximum acid-base distance in aldol condensation reaction between acetone and various aldehydes. It appears that the distance between acidic site and basic site immobilized on mesoporous solid should be an essential factor for catalysis optimization. -- Graphical abstract: Proximal-C-A-SBA-15 with a proximal acid-base distance and maximum-C-A-SBA-15 with a maximum acid-base distance were synthesized by immobilizing lysine onto carboxyl-SBA-15. Display Omitted Research highlights: {yields} Proximal-C-A-SBA-15 with a proximal acid-base distance. {yields} Maximum-C-A-SBA-15 with a maximum acid-base distance. {yields} Compared to maximum-C-A-SBA-15, proximal-C-A-SBA-15 was more active toward aldol condensation reaction between acetone and various aldehydes.

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

    PubMed

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

    2015-03-01

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

  11. A mutational analysis of the active site of human type II inosine 5'-monophosphate dehydrogenase.

    PubMed

    Futer, Olga; Sintchak, Michael D; Caron, Paul R; Nimmesgern, Elmar; DeCenzo, Maureen T; Livingston, David J; Raybuck, Scott A

    2002-01-31

    The oxidation of IMP to XMP is the rate-limiting step in the de novo synthesis of guanine ribonucleotides. This NAD-dependent reaction is catalyzed by the enzyme inosine monophosphate dehydrogenase (IMPDH). Based upon the recent structural determination of IMPDH complexed to oxidized IMP (XMP*) and the potent uncompetitive inhibitor mycophenolic acid (MPA), we have selected active site residues and prepared mutants of human type II IMPDH. The catalytic parameters of these mutants were determined. Mutations G326A, D364A, and the active site nucleophile C331A all abolish enzyme activity to less than 0.1% of wild type. These residues line the IMP binding pocket and are necessary for correct positioning of the substrate, Asp364 serving to anchor the ribose ring of the nucleotide. In the MPA/NAD binding site, significant loss of activity was seen by mutation of any residue of the triad Arg322, Asn303, Asp274 which form a hydrogen bonding network lining one side of this pocket. From a model of NAD bound to the active site consistent with the mutational data, we propose that these resides are important in binding the ribose ring of the nicotinamide substrate. Additionally, mutations in the pair Thr333, Gln441, which lies close to the xanthine ring, cause a significant drop in the catalytic activity of IMPDH. It is proposed that these residues serve to deliver the catalytic water molecule required for hydrolysis of the cysteine-bound XMP* intermediate formed after oxidation by NAD.

  12. A Variable Active Site Residue Influences the Kinetics of Response Regulator Phosphorylation and Dephosphorylation.

    PubMed

    Immormino, Robert M; Silversmith, Ruth E; Bourret, Robert B

    2016-10-04

    Two-component regulatory systems, minimally composed of a sensor kinase and a response regulator protein, are common mediators of signal transduction in microorganisms. All response regulators contain a receiver domain with conserved active site residues that catalyze the signal activating and deactivating phosphorylation and dephosphorylation reactions. We explored the impact of variable active site position T+1 (one residue C-terminal to the conserved Thr/Ser) on reaction kinetics and signaling fidelity, using wild type and mutant Escherichia coli CheY, CheB, and NarL to represent the three major sequence classes observed across response regulators: Ala/Gly, Ser/Thr, and Val/Ile/Met, respectively, at T+1. Biochemical and structural data together suggested that different amino acids at T+1 impacted reaction kinetics by altering access to the active site while not perturbing overall protein structure. A given amino acid at position T+1 had similar effects on autodephosphorylation in each protein background tested, likely by modulating access of the attacking water molecule to the active site. Similarly, rate constants for CheY autophosphorylation with three different small molecule phosphodonors were consistent with the steric constraints on access to the phosphorylation site arising from combination of specific phosphodonors with particular amino acids at T+1. Because other variable active site residues also influence response regulator phosphorylation biochemistry, we began to explore how context (here, the amino acid at T+2) affected the influence of position T+1 on CheY autocatalytic reactions. Finally, position T+1 affected the fidelity and kinetics of phosphotransfer between sensor kinases and response regulators but was not a primary determinant of their interaction.

  13. 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.

  14. Experimental and computational active site mapping as a starting point to fragment-based lead discovery.

    PubMed

    Behnen, Jürgen; Köster, Helene; Neudert, Gerd; Craan, Tobias; Heine, Andreas; Klebe, Gerhard

    2012-02-06

    Small highly soluble probe molecules such as aniline, urea, N-methylurea, 2-bromoacetate, 1,2-propanediol, nitrous oxide, benzamidine, and phenol were soaked into crystals of various proteins to map their binding pockets and to detect hot spots of binding with respect to hydrophobic and hydrophilic properties. The selected probe molecules were first tested at the zinc protease thermolysin. They were then applied to a wider range of proteins such as protein kinase A, D-xylose isomerase, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase, endothiapepsin, and secreted aspartic protease 2. The crystal structures obtained clearly show that the probe molecules populate the protein binding pockets in an ordered fashion. The thus characterized, experimentally observed hot spots of binding were subjected to computational active site mapping using HotspotsX. This approach uses knowledge-based pair potentials to detect favorable binding positions for various atom types. Good agreement between the in silico hot spot predictions and the experimentally observed positions of the polar hydrogen bond forming functional groups and hydrophobic portions was obtained. Finally, we compared the observed poses of the small-molecule probes with those of much larger structurally related ligands. They coincide remarkably well with the larger ligands, considering their spatial orientation and the experienced interaction patterns. This observation confirms the fundamental hypothesis of fragment-based lead discovery: that binding poses, even of very small molecular probes, do not significantly deviate or move once a ligand is grown further into the binding site. This underscores the fact that these probes populate given hot spots and can be regarded as relevant seeds for further design.

  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. Roles of key active-site residues in flavocytochrome P450 BM3.

    PubMed Central

    Noble, M A; Miles, C S; Chapman, S K; Lysek, D A; MacKay, A C; Reid, G A; Hanzlik, R P; Munro, A W

    1999-01-01

    The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 microM, kcat 3960 min-1; Y51F mutant, Km 432 microM, kcat 6140 min-1; wild-type, Km 288 microM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (DeltaG) resulting from a smaller DeltaG of substrate binding. The side chain of Phe-42 acts as a phenyl 'cap' over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2. 08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 microM, kcat 14620 min-1; compared with values of 4.7 microM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a 'fast' to a 'slow' rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat 'fast', 760 (1620) min-1; kcat 'slow', 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl

  17. Functional biomimetic models for the active site in the respiratory enzyme cytochrome c oxidase.

    PubMed

    Collman, James P; Decréau, Richard A

    2008-11-07

    A functional analog of the active site in the respiratory enzyme, cytochrome c oxidase (CcO) reproduces every feature in CcO's active site: a myoglobin-like heme (heme a3), a distal tridentate imidazole copper complex (Cu(B)), a phenol (Tyr244), and a proximal imidazole. When covalently attached to a liquid-crystalline SAM film on an Au electrode, this functional model continuously catalyzes the selective four-electron reduction of dioxygen at physiological potential and pH, under rate-limiting electron flux (as occurs in CcO).

  18. Antimicrobial activity and phenolic content of natural site and micropropagated Limonium avei (De Not.) Brullo & Erben plant extracts.

    PubMed

    Nostro, Antonia; Filocamo, Angela; Giovannini, Annalisa; Catania, Stefania; Costa, Chiara; Marino, Andreana; Bisignano, Giuseppe

    2012-01-01

    This study reported the antimicrobial activity and phenolic content of natural site and micropropagated Limonium avei (De Not.) Brullo & Erben inflorescences. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ethanolic extracts were determined according to the Clinical Laboratory Standards Institute guidelines. Individual phenolic acids and flavonoids were detected by a high performance liquid chromatography (HPLC-DAD) method. The samples showed a comparable antimicrobial activity, although the natural site extract possessed the lower MIC values. The best activity was detected against Gram-positive bacteria, such as Staphylococcus aureus including methicillin resistant strains (MIC and MBC values ranging from 7.81 to 62.50 µg mL(-1) and from 500 to 2000 µg mL(-1) respectively). In contrast, a low activity was found on Gram-negative bacteria and Candida albicans. The HPLC-DAD analysis revealed ten phenolic acids and four flavonoids with a major amount of m-coumaric acid, naringin and quercetin in the natural site extract.

  19. 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

  20. 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

  1. Healing of complement activating Ti implants compared with non-activating Ti in rat tibia.

    PubMed

    Harmankaya, N; Igawa, K; Stenlund, P; Palmquist, A; Tengvall, P

    2012-09-01

    Recent studies have revealed that ozone ultraviolet (UVO) illumination of titanium (Ti) implants improves bone-implant anchorage by altering the physico-chemical and immune activating properties of the titanium dioxide (TiO(2)) layer. In the present rat tibia model, the authors compared the early events of inflammation and bone formation around UVO-treated Ti and complement activating immunoglobin g (IgG)-coated Ti. Machined Ti and machined Ti coated with a physical vapour-deposited Ti layer were used as references. Screw-shaped test and reference implants were implanted into rat tibia and harvested after 1, 7 and 28 days. Messenger RNA expression of implant adhered cells and peri-implant tissue ~250 μm from the surface were subsequently analysed with regard to IL-1β, TNF-α, osteocalcin, cathepsin K, BMP-2 and PDGF. Separate implants were retrieved after 7 and 28 days for removal torque measurements, and histological staining and histomorphometric analysis of bone area and bone-to-implant contact. While enhanced expression of inflammatory markers, TNF-α and IL-1β, was observed on IgG-coated surfaces throughout the observation time, UVO-treated surfaces indicated a significantly lower early inflammatory response. In the early phases (1 and 7 days), the UVO-treated surfaces displayed a significantly higher expression of osteoblast markers BMP-2 and osteocalcin. In summary, complement activating Ti implants elicited a stronger inflammatory response than UVO-treated Ti, with low complement activation during the first week of healing. In spite of this, the UVO-treated Ti induced only marginally more bone growth outside the implants.

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

    PubMed

    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.

  3. Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites.

    PubMed

    Gu, Yuanzheng; Barry, Joshua; Gu, Chen

    2013-05-15

    Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.

  4. 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

  5. Tuned by metals: the TET peptidase activity is controlled by 3 metal binding sites.

    PubMed

    Colombo, Matteo; Girard, Eric; Franzetti, Bruno

    2016-02-08

    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 Mn(2+), hyperthermophilic TETs prefers Co(2+). 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.

  6. Fragile Sites of 'Valencia' Sweet Orange (Citrus sinensis) Chromosomes Are Related with Active 45s rDNA.

    PubMed

    Lan, Hong; Chen, Chun-Li; Miao, Yin; Yu, Chang-Xiu; Guo, Wen-Wu; Xu, Qiang; Deng, Xiu-Xin

    2016-01-01

    Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in 'Valencia' C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of 'Valencia' C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid 'Valencia' C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in 'Valencia' sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in 'Valencia' sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites.

  7. Site- and horizon-specific patterns of microbial community structure and enzyme activities in permafrost-affected soils of Greenland

    PubMed Central

    Gittel, Antje; Bárta, Jiří; Kohoutová, Iva; Schnecker, Jörg; Wild, Birgit; Čapek, Petr; Kaiser, Christina; Torsvik, Vigdis L.; Richter, Andreas; Schleper, Christa; Urich, Tim

    2014-01-01

    Permafrost-affected soils in the Northern latitudes store huge amounts of organic carbon (OC) that is prone to microbial degradation and subsequent release of greenhouse gasses to the atmosphere. In Greenland, the consequences of permafrost thaw have only recently been addressed, and predictions on its impact on the carbon budget are thus still highly uncertain. However, the fate of OC is not only determined by abiotic factors, but closely tied to microbial activity. We investigated eight soil profiles in northeast Greenland comprising two sites with typical tundra vegetation and one wet fen site. We assessed microbial community structure and diversity (SSU rRNA gene tag sequencing, quantification of bacteria, archaea and fungi), and measured hydrolytic and oxidative enzyme activities. Sampling site and thus abiotic factors had a significant impact on microbial community structure, diversity and activity, the wet fen site exhibiting higher potential enzyme activities and presumably being a hot spot for anaerobic degradation processes such as fermentation and methanogenesis. Lowest fungal to bacterial ratios were found in topsoils that had been relocated by cryoturbation (“buried topsoils”), resulting from a decrease in fungal abundance compared to recent (“unburied”) topsoils. Actinobacteria (in particular Intrasporangiaceae) accounted for a major fraction of the microbial community in buried topsoils, but were only of minor abundance in all other soil horizons. It was indicated that the distribution pattern of Actinobacteria and a variety of other bacterial classes was related to the activity of phenol oxidases and peroxidases supporting the hypothesis that bacteria might resume the role of fungi in oxidative enzyme production and degradation of phenolic and other complex substrates in these soils. Our study sheds light on the highly diverse, but poorly-studied communities in permafrost-affected soils in Greenland and their role in OC degradation. PMID

  8. Site- and horizon-specific patterns of microbial community structure and enzyme activities in permafrost-affected soils of Greenland.

    PubMed

    Gittel, Antje; Bárta, Jiří; Kohoutová, Iva; Schnecker, Jörg; Wild, Birgit; Capek, Petr; Kaiser, Christina; Torsvik, Vigdis L; Richter, Andreas; Schleper, Christa; Urich, Tim

    2014-01-01

    Permafrost-affected soils in the Northern latitudes store huge amounts of organic carbon (OC) that is prone to microbial degradation and subsequent release of greenhouse gasses to the atmosphere. In Greenland, the consequences of permafrost thaw have only recently been addressed, and predictions on its impact on the carbon budget are thus still highly uncertain. However, the fate of OC is not only determined by abiotic factors, but closely tied to microbial activity. We investigated eight soil profiles in northeast Greenland comprising two sites with typical tundra vegetation and one wet fen site. We assessed microbial community structure and diversity (SSU rRNA gene tag sequencing, quantification of bacteria, archaea and fungi), and measured hydrolytic and oxidative enzyme activities. Sampling site and thus abiotic factors had a significant impact on microbial community structure, diversity and activity, the wet fen site exhibiting higher potential enzyme activities and presumably being a hot spot for anaerobic degradation processes such as fermentation and methanogenesis. Lowest fungal to bacterial ratios were found in topsoils that had been relocated by cryoturbation ("buried topsoils"), resulting from a decrease in fungal abundance compared to recent ("unburied") topsoils. Actinobacteria (in particular Intrasporangiaceae) accounted for a major fraction of the microbial community in buried topsoils, but were only of minor abundance in all other soil horizons. It was indicated that the distribution pattern of Actinobacteria and a variety of other bacterial classes was related to the activity of phenol oxidases and peroxidases supporting the hypothesis that bacteria might resume the role of fungi in oxidative enzyme production and degradation of phenolic and other complex substrates in these soils. Our study sheds light on the highly diverse, but poorly-studied communities in permafrost-affected soils in Greenland and their role in OC degradation.

  9. Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites*

    PubMed Central

    Honaker, Matthew T.; Acchione, Mauro; Zhang, Wei; Mannervik, Bengt; Atkins, William M.

    2013-01-01

    The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes. PMID:23649628

  10. 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.

  11. Active layer dynamics in three sites with contrasted topography in the Byers Peninsula (Livingston Island, Antarctica)

    NASA Astrophysics Data System (ADS)

    Oliva, Marc; Ruiz-Fernández, Jesús; Vieira, Gonçalo

    2015-04-01

    Topography exerts a key role on permafrost distribution in areas where mean annual temperatures are slightly negative. This is the case of low-altitude environments in Maritime Antarctica, namely in the South Shetland Islands, where permafrost is marginal to discontinuous until elevations of 20-40 m asl turning to continuous at higher areas. Consequently, the active layer dynamics is also strongly conditioned by the geomorphological setting. In January 2014 we installed three sites for monitoring the active layer dynamics across the Byers Peninsula (Livingston Island, South Shetland Islands) in different geomorphological environments at elevations between 60 and 100 m. The purpose was to examine the role of the topography and microclimatic conditions on the active layer dynamics. At each site a set of loggers was set up to monitor: air temperatures, snow thickness, ground temperatures until 80 cm together with the coupling atmosphere-ground temperatures. During the first year of monitoring the mean annual air temperatures show similar values in the three sites, in all cases slightly below freezing. The snowy conditions during this year in this archipelago have resulted in a late melting of snow, which has also conditioned the duration of frozen conditions in the uppermost soil layers. Topography has a strong influence on snow cover duration, which in turn affects frozen ground conditions. The Domo site is located in a higher position with respect to the central plateau of Byers; here, the wind is stronger and snow cover thinner, which has conditioned a longer thawing season than in the other two sites (Cerro Negro, Escondido). These two sites are located in topographically protected areas favouring snow accumulation. The longer persistence of snow conditions a longer duration of negative temperatures in the active layer of the permafrost. This research was financially supported by the HOLOANTAR project (Portuguese Science Foundation) and the AXA Research Fund.

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

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-05

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF THE INTERIOR Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the Atlantic... Notice of Intent to Prepare an Environmental Assessment (EA) for Commercial Wind Leasing and...

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

    ERIC Educational Resources Information Center

    Rottmann, Cindy

    2008-01-01

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

  14. 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

  15. 77 FR 5830 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-06

    ... Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the Atlantic... of involving Federal agencies, states, tribes, local governments, offshore wind energy developers, and the public in the Department of the Interior's (DOI) ``Smart from the Start'' wind...

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

    PubMed

    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

    2016-09-01

    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.

  17. The active site of cytochrome P-450 nifedipine oxidase: a model-building study.

    PubMed

    Ferenczy, G G; Morris, G M

    1989-12-01

    A model of the active site of cytochrome P-450 nifedipine oxidase is built on the basis of sequence homology with cytochrome P-450CAM. Substrates are docked into the binding pocket, and molecular mechanical energy minimization is performed to analyze the forces between the substrates and the enzyme.

  18. 77 FR 74218 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-13

    ... identified in the document entitled, Commercial Leasing for Wind Power on the Outer Continental Shelf... Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the Atlantic... agencies, states, tribes, local governments, offshore wind energy developers, and the public in...

  19. ACCELERATED SITE TECHNOLOGY DEPLOYMENT COST AND PERFORMANCE REPORT COMPARABILITY OF ISOCS INSTRUMENT IN RADIONUCLIDE CHARACTERICATION AT BROOKHAVEN NATIONAL LABORATORY

    SciTech Connect

    KALB,P.; LUCKETT,L.; MILLER,K.; GOGOLAK,C.; MILIAN,L.

    2001-03-01

    This report describes a DOE Accelerated Site Technology Deployment project being conducted at Brookhaven National Laboratory to deploy innovative, radiological, in situ analytical techniques. The technologies are being deployed in support of efforts to characterize the Brookhaven Graphite Research Reactor (BGRR) facility, which is currently undergoing decontamination and decommissioning. This report focuses on the deployment of the Canberra Industries In Situ Object Counting System (ISOCS) and assesses its data comparability to baseline methods of sampling and laboratory analysis. The battery-operated, field deployable gamma spectrometer provides traditional spectra of counts as a function of gamma energy. The spectra are then converted to radionuclide concentration by applying innovative efficiency calculations using monte carlo statistical methods and pre-defined geometry templates in the analysis software. Measurement of gamma emitting radionuclides has been accomplished during characterization of several BGRR components including the Pile Fan Sump, Above Ground Ducts, contaminated cooling fans, and graphite pile internals. Cs-137 is the predominant gamma-emitting radionuclide identified, with smaller quantities of Co-60 and Am-241 detected. The Project used the Multi-Agency Radiation Survey and Site Investigation Manual guidance and the Data Quality Objectives process to provide direction for survey planning and data quality assessment. Analytical results have been used to calculate data quality indicators (DQI) for the ISOCS measurements. Among the DQIs assessed in the report are sensitivity, accuracy, precision, bias, and minimum detectable concentration. The assessment of the in situ data quality using the DQIs demonstrates that the ISOCS data quality can be comparable to definitive level laboratory analysis when the field instrument is supported by an appropriate Quality Assurance Project Plan. A discussion of the results obtained by ISOCS analysis of

  20. 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.

  1. Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

    PubMed Central

    Moomaw, Ellen W.; Hoffer, Eric; Moussatche, Patricia; Salerno, John C.; Grant, Morgan; Immelman, Bridget; Uberto, Richard; Ozarowski, Andrew; Angerhofer, Alexander

    2013-01-01

    Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site. PMID:23469254

  2. Dynamics of an Active-Site Flap Contributes to Catalysis in a JAMM Family Metallo Deubiquitinase.

    PubMed

    Bueno, Amy N; Shrestha, Rashmi K; Ronau, Judith A; Babar, Aditya; Sheedlo, Michael J; Fuchs, Julian E; Paul, Lake N; Das, Chittaranjan

    2015-10-06

    The endosome-associated deubiquitinase (DUB) AMSH is a member of the JAMM family of zinc-dependent metallo isopeptidases with high selectivity for Lys63-linked polyubiquitin chains, which play a key role in endosomal-lysosomal sorting of activated cell surface receptors. The catalytic domain of the enzyme features a flexible flap near the active site that opens and closes during its catalytic cycle. Structural analysis of its homologues, AMSH-LP (AMSH-like protein) and the fission yeast counterpart, Sst2, suggests that a conserved Phe residue in the flap may be critical for substrate binding and/or catalysis. To gain insight into the contribution of this flap in substrate recognition and catalysis, we generated mutants of Sst2 and characterized them using a combination of enzyme kinetics, X-ray crystallography, molecular dynamics simulations, and isothermal titration calorimetry (ITC). Our analysis shows that the Phe residue in the flap contributes key interactions during the rate-limiting step but not to substrate binding, since mutants of Phe403 exhibit a defect only in kcat but not in KM. Moreover, ITC studies show Phe403 mutants have similar KD for ubiquitin compared to the wild-type enzyme. The X-ray structures of both Phe403Ala and the Phe403Trp, in both the free and ubiquitin bound form, reveal no appreciable structural change that might impair substrate or alter product binding. We observed that the side chain of the Trp residue is oriented identically with respect to the isopeptide moiety of the substrate as the Phe residue in the wild-type enzyme, so the loss of activity seen in this mutant cannot be explained by the absence of a group with the ability to provide van der Waals interactions that facilitate the hyrdolysis of the Lys63-linked diubiquitin. Molecular dynamics simulations indicate that the flap in the Trp mutant is quite flexible, allowing almost free rotation of the indole side chain. Therefore, it is possible that these different dynamic

  3. Transfer hydrogenation over sodium-modified ceria: Enrichment of redox sites active for alcohol dehydrogenation

    DOE PAGES

    Nelson, Nicholas C.; Boote, Brett W.; Naik, Pranjali; ...

    2017-01-17

    Ceria (CeO2) and sodium-modified ceria (Ce-Na) were prepared through combustion synthesis. Palladium was deposited onto the supports (Pd/CeO2 and Pd/Ce-Na) and their activity for the aqueous-phase transfer hydrogenation of phenol using 2-propanol under liquid flow conditions was studied. Pd/Ce-Na showed a marked increase (6×) in transfer hydrogenation activity over Pd/CeO2. Material characterization indicated that water-stable sodium species were not doped into the ceria lattice, but rather existed as subsurface carbonates. Modification of ceria by sodium provided more adsorption and redox active sites (i.e. defects) for 2-propanol dehydrogenation. This effect was an intrinsic property of the Ce-Na support and independent ofmore » Pd. The redox sites active for 2-propanol dehydrogenation were thermodynamically equivalent on both supports/catalysts. At high phenol concentrations, the reaction was limited by 2-propanol adsorption. Furthermore, the difference in catalytic activity was attributed to the different numbers of 2-propanol adsorption and redox active sites on each catalyst.« less

  4. Comparative Plutonium-239 Dose Assessment for Three Desert Sites: Maralinga, Australia; Palomares, Spain; and the Nevada Test Site, USA - Before and After Remedial Action

    SciTech Connect

    Church, B W; Shinn, J; Williams, G A; Martin, L J; O'Brien, R S; Adams, S R

    2000-07-14

    As a result of nuclear weapons testing and accidents, plutonium has been distributed into the environment. The areas close to the sites of these tests and accidental dispersions contain plutonium deposition of such a magnitude that health authorities and responsible officials have mandated that the contaminated areas be protected, generally through isolation or removal of the contaminated areas. In recent years remedial actions have taken place at all these sites. For reasons not entirely clear, the public perceives radiation exposure risk to be much greater than the evidence would suggest [1]. This perception seems to be particularly true for plutonium, which has often been ''demonized'' in various publications as the ''most hazardous substance known to man'' [2]. As the position statement adapted by the Health Physics Society explains, ''Plutonium's demonization is an example of how the public has been misled about radiation's environmental and health threats generally, and in cases like plutonium, how it has developed a warped ''risk perception'' that does not reflect reality'' [3]. As a result of this risk perception and ongoing debate surrounding environmental plutonium contamination, remedial action criteria are difficult to establish. By examining the data available before and after remedial actions taken at the three sites discussed in our report, we hope to present data that will illustrate that plutonium measured as aged deposition (older than several months) does not present as high a dose or risk as many had expected. The authors show that even though dose to the lung from inhalation (the primary pathway for the high-fired plutonium oxide particles present at these sites) is reduced, such a reduction is achieved at significant cost. The cost comes from damage to the environment, large expenditures per hectare rehabilitated, and the risk to occupational workers. This paper specifically examines sites that are similar in many ways. These sites were chosen

  5. The controlled relay of multiple protons required at the active site of nitrogenase.

    PubMed

    Dance, Ian

    2012-07-07

    The enzyme nitrogenase, when reducing natural and unnatural substrates, requires large numbers of protons per chemical catalytic cycle. The active face of the catalytic site (the FeMo-cofactor, FeMo-co) is situated in a protein domain which is largely hydrophobic and anhydrous, and incapable of serial provision of multiple protons. Through detailed analysis of the high quality protein crystal structures available the characteristics of a chain of water molecules leading from the protein surface to a key sulfur atom (S3B) of FeMo-co are described. The first half of the water chain from the surface inwards is branched, slightly variable, and able to accommodate exogenous small molecules: this is dubbed the proton bay. The second half, from the proton bay to S3B, is comprised of a single chain of eight hydrogen bonded water molecules. This section is strictly conserved, and is intimately involved in hydrogen bonds with homocitrate, an essential component that chelates Mo. This is the proton wire, and a detailed Grotthuss mechanism for serial translocation of protons through this proton wire to S3B is proposed. This controlled serial proton relay from the protein surface to S3B is an essential component of the intramolecular hydrogenation paradigm for the complete chemical mechanisms of nitrogenase. Each proton reaching S3B, instigated by electron transfer to FeMo-co, becomes a hydrogen atom that migrates to other components of the active face of FeMo-co and to bound substrates and intermediates, allowing subsequent multiple proton transfers along the proton wire. Experiments to test the proposed mechanism of proton supply are suggested. The water chain in nitrogenase is comparable with the purported proton pumping pathway of cytochrome c oxidase.

  6. Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

    PubMed Central

    Davidge, Kelly S; Singh, Sandip; Bowman, Lesley AH; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radi, Rafael; Poole, Robert K; Dikshit, Kanak; Estrin, Dario A; Marti, Marcelo A; Boechi, Leonardo

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and •NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify •NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, •NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site. PMID:26478812

  7. Site-specific profiles of estrogenic activity in agricultural areas of California's inland waters.

    PubMed

    Lavado, Ramon; Loyo-Rosales, Jorge E; Floyd, Emily; Kolodziej, Edward P; Snyder, Shane A; Sedlak, David L; Schlenk, Daniel

    2009-12-15

    To evaluate the occurrence and sources of compounds capable of feminizing fish in agriculturally impacted waterways of the Central Valley of California, water samples were extracted and subjected to chemical analyses as well as in vitro and in vivo measurements of vitellogenin in juvenile rainbow trout (Oncorhynchus mykiss). Among the 16 sites sampled, 6 locations frequently exhibited elevated concentrations of estrogenic substances with 17beta-estradiol equivalents up to 242 ng/L in vitro and 12 microg/kg in vivo. The patterns of activity varied among sites, with two sites showing elevated activity only in vitro, two showing elevated activity only in vivo, and two showing elevated activity in both assays. Sequential elution of solid-phase extraction (SPE) disks followed by bioassay-guided fractionation was used to characterize water samples from the two locations where activity was observed in both bioassays. The highest estrogenic activity was observed in the most nonpolar fractions (80-100% methanol eluent) from the Napa River, while most of the activity in the Sacramento River Delta eluted in the 60% methanol eluent. Quantitative analyses of SPE extracts and additional HPLC fractionation of the SPE extracts by GC-MS/MS and LC-MS/MS indicated concentrations of steroid hormones, alkylphenol polyethoxylates, and herbicides that were at least 1-3 orders of magnitude below bioassay 17beta-estradiol equivalent calculations. Given the different patterns of activity and chemical properties of the estrogenic compounds, it appears that estrogenic activity in these agriculturally impacted surface waters is attributable to multiple compounds. Further investigation is needed to identify the compounds causing the estrogenic activity and to determine the potential impacts of these compounds on feral fish.

  8. Superior long-term activity for a Pt-Re alloy compared to Pt in methanol oxidation reactions

    NASA Astrophysics Data System (ADS)

    Duke, Audrey S.; Xie, Kangmin; Monnier, John R.; Chen, Donna A.

    2017-03-01

    Pt-Re bimetallic catalysts have shown enhanced activity compared to pure Pt for reactions involving oxidation, but the origins of this improved activity are not fully understood. Methanol oxidation on a Pt-Re alloy surface and pure Pt foil was studied in a microreactor coupled to an ultrahigh vacuum chamber. For reaction at 60 °C, the Pt-Re alloy surface exhibits superior long-term activity over a 24 h reaction period compared to pure Pt. The initial activity of Pt is 10-15% higher than on Pt-Re; however, the Pt surface gradually loses activity after 10 h online, whereas the activity of Pt-Re does not diminish. Post-reaction XPS shows that more carbon accumulates on the Pt than on Pt-Re, and the improved long-term activity is attributed to a greater ability of Pt-Re to oxidize the carbonaceous intermediates that eventually poison active sites. Both Pt and Pt-Re surfaces have almost no activity for methanol oxidation until a minimum coverage of oxygen is achieved from O2 dissociation. A comparison with methanol oxidation studies on Pt and Pt-Re in a pressure regime that is 150 times lower than in this work demonstrates that more carbon and less oxygen accumulate on the surfaces during reaction at the lower pressures.

  9. Site-directed mutagenesis of an alkaline phytase: influencing specificity, activity and stability in acidic milieu.

    PubMed

    Tran, Thuy T; Mamo, Gashaw; Búxo, Laura; Le, Nhi N; Gaber, Yasser; Mattiasson, Bo; Hatti-Kaul, Rajni

    2011-07-10

    Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.

  10. Structure of Arabidopsis thaliana 5-methylthioribose kinase reveals a more occluded active site than its bacterial homolog

    PubMed Central

    Ku, Shao-Yang; Cornell, Kenneth A; Howell, P Lynne

    2007-01-01

    Background Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics. Results The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATPγS and MTR has been determined at 1.9 Å resolution. The structure is similar to B. subtilis MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of A. thaliana and B. subtilis MTR kinase adopt different conformations despite high sequence similarity. The ATPγS analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the A. thaliana enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR. Conclusion The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase. PMID:17961230

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

    SciTech Connect

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

    2007-01-01

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

  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. Multiple DNA binding activities of the novel site-specific recombinase, Piv, from Moraxella lacunata.

    PubMed

    Tobiason, D M; Lenich, A G; Glasgow, A C

    1999-04-02

    The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment in Moraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the lambda Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region of M. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome.

  14. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

    PubMed

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-08

    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.

  15. 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

  16. 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.

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

    PubMed

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

    2015-01-01

    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.

  18. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase.

    PubMed

    Irani, Seema; Yogesha, S D; Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L; Nie, Grace; Prescott, Nicholas A; Zhang, Yan Jessie

    2016-03-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.

  19. Mutational analysis of the lac regulatory region: second-site changes that activate mutant promoters.

    PubMed Central

    Rothmel, R K; LeClerc, J E

    1989-01-01

    Second-site mutations that restored activity to severe lacP1 down-promoter mutants were isolated. This was accomplished by using a bacteriophage f1 vector containing a fusion of the mutant E. coli lac promoters with the structural gene for chloramphenicol acetyltransferase (CAT), so that a system was provided for selecting phage revertants (or pseudorevertants) that conferred resistance of phage-infected cells to chloramphenicol. Among the second-site changes that relieved defects in mutant lac promoters, the only one that restored lacP1 activity was a T----G substitution at position -14, a weakly conserved site in E. coli promoters. Three other sequence changes, G----A at -2, A----T at +1, and C----A at +10, activated nascent promoters in the lac regulatory region. The nascent promoters conformed to the consensus rule, that activity is gained by sequence changes toward homology with consensus sequences at the -35 and -10 regions of the promoter. However, the relative activities of some promoters cannot be explained solely by consideration of their conserved sequence elements. Images PMID:2660105

  20. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase

    PubMed Central

    Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L.; Nie, Grace; Prescott, Nicholas A.; Zhang, Yan Jessie

    2016-01-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families. PMID:26933063

  1. 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

  2. Risks to children from exposure to lead in air during remedial or removal activities at Superfund sites: a case study of the RSR lead smelter Superfund site.

    PubMed

    Khoury, Ghassan A; Diamond, Gary L

    2003-01-01

    at the downwind West Dallas community did not result in more than 5% of children exceeding the target blood lead levels. The models were also used to estimate air lead levels for short-term and long-term exposures that would not exceed specified levels of risk (risk-based concentrations, RBCs). RBCs were derived for various daily exposure durations (3 or 8 h/day) and frequencies (1-7 days/week). RBCs based on the ICRP model ranged from 0.3 (7 days/week, 8 h/day) to 4.4 microg/m(3) (1 day/week, 3 h/day) for long-term exposures and were lower than those based on the IEUBK model. For short-term exposures, the RBCs ranged from 3.5 to 29.0 microg/m(3). Recontamination of remediated residential yards from deposition of air lead emitted during remedial activities at the RSR Superfund site was also examined. The predicted increase in soil concentration due to lead deposition at the monitoring station, which represented the community at large, was 3.0 mg/kg. This potential increase in soil lead concentration was insignificant, less than 1% increase, when compared to the clean-up level of 500 mg/kg developed for residential yards at the site.

  3. Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents.

    PubMed

    Reddi, Ravikumar; Arya, Tarun; Kishor, Chandan; Gumpena, Rajesh; Ganji, Roopa J; Bhukya, Supriya; Addlagatta, Anthony

    2014-09-01

    Methionine aminopeptidases (MetAPs) cleave initiator methionine from ~ 70% of the newly synthesized proteins in every living cell, and specific inhibition or knockdown of this function is detrimental. MetAPs are metalloenzymes, and are broadly classified into two subtypes, type I and type II. Bacteria contain only type I MetAPs, and the active site of these enzymes contains a conserved cysteine. By contrast, in type II enzymes the analogous position is occupied by a conserved glycine. Here, we report the reactivity of the active site cysteine in a type I MetAP, MetAP1c, of Mycobacterium tuberculosis (MtMetAP1c) towards highly selective cysteine-specific reagents. The authenticity of selective modification of Cys105 of MtMetAP1c was established by using site-directed mutagenesis and crystal structure determination of covalent and noncovalent complexes. On the basis of these observations, we propose that metal ions in the active site assist in the covalent modification of Cys105 by orienting the reagents appropriately for a successful reaction. These studies establish, for the first time, that the conserved cysteine of type I MetAPs can be targeted for selective inhibition, and we believe that this chemistry can be exploited for further drug discovery efforts regarding microbial MetAPs.

  4. Computational approaches to find the active binding sites of biological targets against busulfan.

    PubMed

    Karthick, T; Tandon, Poonam

    2016-06-01

    Determination of electrophilic and nucleophilic sites of a molecule is the primary task to find the active sites of the lead molecule. In the present study, the active sites of busulfan have been predicted by molecular electrostatic potential surface and Fukui function analysis with the help of dispersion corrected density functional theory. Similarly, the identification of active binding sites of the proteins against lead compound plays a vital role in the field of drug discovery. Rigid and flexible molecular docking approaches are used for this purpose. For rigid docking, Hex 8.0.0 software employing fast Fourier transform (FFT) algorithm has been used. The partial flexible blind docking simulations have been performed with AutoDock 4.2 software; where a Lamarckian genetic algorithm is employed. The results showed that the most electrophilic atoms of busulfan bind with the targets. It is clear from the docking studies that busulfan has inhibition capability toward the targets 12CA and 1BZM. Graphical Abstract Docking of ligand and protein.

  5. 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.

  6. 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.

  7. Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis.

    PubMed

    Roche, Eric D; Walsh, Christopher T

    2003-02-11

    Nonribosomal peptide synthetases (NRPSs) make many natural products of clinical importance, but a deeper understanding of the protein domains that compose NRPS assembly lines is required before these megasynthetases can be effectively engineered to produce novel drugs. The N-terminal amide bond-forming condensation (C) domain of the enterobactin NRPS EntF was excised from the multidomain synthetase using endpoints determined from sequence alignments and secondary structure predictions. The isolated domain was well-folded when compared by circular dichroism to the vibriobactin NRPS VibH, a naturally free-standing C domain. The EntF domain was also fully functional in an assay based on a synthetic small-molecule substrate, seryl N-acetylcysteamine. Active site mutants of the EntF C domain were surprisingly inactive in vitro as compared to their VibH counterparts, yet maintained the overall domain structure. An in vivo assay was developed in the context of the full-length EntF protein to more sensitively probe the activity level of the C domain mutants, and this supported strong effects for the active site mutations. The crucial role of histidine-138 was confirmed by assay of the full-length protein in vitro. These results suggest a strong resemblance of catalysis by the EntF C domain to chloramphenicol acetyltransferase, including an active site organized by an arginine-aspartate salt bridge, a key histidine acting as a general base, and an asparagine instead of a serine stabilizing the proposed tetrahedral intermediate by hydrogen bonding. The precise definition of a functional C domain excised from a NRPS should aid efforts at swapping NRPS domains between assembly lines.

  8. Technical basis for classification of low-activity waste fraction from Hanford site tanks

    SciTech Connect

    Petersen, C.A., Westinghouse Hanford

    1996-07-17

    The overall objective of this report is to provide a technical basis to support a U.S. Nuclear Regulatory Commission determination to classify the low-activity waste from the Hanford Site single-shell and double-shell tanks as `incidental` wastes after removal of additional radionuclides and immobilization.The proposed processing method, in addition to the previous radionuclide removal efforts, will remove the largest practical amount of total site radioactivity, attributable to high-level wastes, for disposal in a deep geologic repository. The remainder of the waste would be considered `incidental` waste and could be disposed onsite.

  9. Technical basis for classification of low-activity waste fraction from Hanford site tanks

    SciTech Connect

    Petersen, C.A.

    1996-09-20

    The overall objective of this report is to provide a technical basis to support a U.S. Nuclear Regulatory Commission determination to classify the low-activity waste from the Hanford Site single-shell and double-shell tanks as `incidental` wastes after removal of additional radionuclides and immobilization.The proposed processing method, in addition to the previous radionuclide removal efforts, will remove the largest practical amount of total site radioactivity, attributable to high-level waste, for disposal is a deep geologic repository. The remainder of the waste would be considered `incidental` waste and could be disposed onsite.

  10. Particle Size Distribution Data From Existing Boreholes at the Immobilized Low-Activity Waste Site

    SciTech Connect

    Valenta, Michelle M.; Martin, Maria B.; Moreno, Jorge R.; Ferri, Rosalie M.; Horton, Duane G.; Reidel, Stephen P.

    2000-09-25

    This report provides particle size distribution data for samples near the Immobilized Low-Activity Waste (ILAW) Site that were archived in the Hanford Geotechnical Sample Library. Seventy-nine sediment samples were analyzed from four boreholes. Samples were collected from every ten feet in the boreholes. Eightly percent of the samples were classified as slightly gravelly sand. Fifteen percent were classified as gravelly sand, gravelly silty sand, or sandy gravels. These data indicate that the particle size of the sediment is consistent across the ILAW site and is dominated by sand in the upper part of the Hanford formation with more gravel rich units in the lower part.

  11. 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.

  12. Understanding the conformational flexibility and electrostatic properties of curcumin in the active site of rhAChE via molecular docking, molecular dynamics, and charge density analysis.

    PubMed

    Saravanan, Kandasamy; Kalaiarasi, Chinnasamy; Kumaradhas, Poomani

    2017-01-04

    Acetylcholinesterase (AChE) is an important enzyme responsible for Alzheimer's disease, as per report, keto-enol form of curcumin inhibits this enzyme. The present study aims to understand the binding mechanism of keto-enol curcumin with the recombinant human Acetylcholinesterase (rhAChE) from its conformational flexibility, intermolecular interactions, charge density distribution, and the electrostatic properties at the active site of rhAChE. To accomplish this, a molecular docking analysis of curcumin with the rhAChE was performed, which gives the structure and conformation of curcumin in the active site of rhAChE. Further, the charge density distribution and the electrostatic properties of curcumin molecule (lifted from the active site of rhAChE) were determined from the high level density functional theory (DFT) calculations coupled with the charge density analysis. On the other hand, the curcumin molecule was optimized (gas phase) using DFT method and further, the structure and charge density analysis were also carried out. On comparing the conformation, charge density distribution and the electrostatic potential of the active site form of curcumin with the corresponding gas phase form reveals that the above said properties are significantly altered when curcumin is present in the active site of rhAChE. The conformational stability and the interaction of curcumin in the active site are also studied using molecular dynamics simulation, which shows a large variation in the conformational geometry of curcumin as well as the intermolecular interactions.

  13. Controlling activation site density by low-energy far-field stimulation in cardiac tissue.

    PubMed

    Hörning, Marcel; Takagi, Seiji; Yoshikawa, Kenichi

    2012-06-01

    Tachycardia and fibrillation are potentially fatal arrhythmias associated with the formation of rotating spiral waves in the heart. Presently, the termination of these types of arrhythmia is achieved by use of antitachycardia pacing or cardioversion. However, these techniques have serious drawbacks, in that they either have limited application or produce undesirable side effects. Low-energy far-field stimulation has recently been proposed as a superior therapy. This proposed therapeutic method would exploit the phenomenon in which the application of low-energy far-field shocks induces a large number of activation sites ("virtual electrodes") in tissue. It has been found that the formation of such sites can lead to the termination of undesired states in the heart and the restoration of normal beating. In this study we investigate a particular aspect of this method. Here we seek to determine how the activation site density depends on the applied electric field through in vitro experiments carried out on neonatal rat cardiac tissue cultures. The results indicate that the activation site density increases exponentially as a function of the intracellular conductivity and the level of cell isotropy. Additionally, we report numerical results obtained from bidomain simulations of the Beeler-Reuter model that are quantitatively consistent with our experimental results. Also, we derive an intuitive analytical framework that describes the activation site density and provides useful information for determining the ratio of longitudinal to transverse conductivity in a cardiac tissue culture. The results obtained here should be useful in the development of an actual therapeutic method based on low-energy far-field pacing. In addition, they provide a deeper understanding of the intrinsic properties of cardiac cells.

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

    SciTech Connect

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

    2011-08-16

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

  15. Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

    PubMed

    Armstrong, Michelle; van Hoorebeke, Christopher; Horn, Thomas; Deschamps, Joshua; Freedman, J Cody; Kalyanaraman, Chakrapani; Jacobson, Matthew P; Holman, Theodore

    2016-11-01

    Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors.

  16. Controlling activation site density by low-energy far-field stimulation in cardiac tissue

    NASA Astrophysics Data System (ADS)

    Hörning, Marcel; Takagi, Seiji; Yoshikawa, Kenichi

    2012-06-01

    Tachycardia and fibrillation are potentially fatal arrhythmias associated with the formation of rotating spiral waves in the heart. Presently, the termination of these types of arrhythmia is achieved by use of antitachycardia pacing or cardioversion. However, these techniques have serious drawbacks, in that they either have limited application or produce undesirable side effects. Low-energy far-field stimulation has recently been proposed as a superior therapy. This proposed therapeutic method would exploit the phenomenon in which the application of low-energy far-field shocks induces a large number of activation sites (“virtual electrodes”) in tissue. It has been found that the formation of such sites can lead to the termination of undesired states in the heart and the restoration of normal beating. In this study we investigate a particular aspect of this method. Here we seek to determine how the activation site density depends on the applied electric field through in vitro experiments carried out on neonatal rat cardiac tissue cultures. The results indicate that the activation site density increases exponentially as a function of the intracellular conductivity and the level of cell isotropy. Additionally, we report numerical results obtained from bidomain simulations of the Beeler-Reuter model that are quantitatively consistent with our experimental results. Also, we derive an intuitive analytical framework that describes the activation site density and provides useful information for determining the ratio of longitudinal to transverse conductivity in a cardiac tissue culture. The results obtained here should be useful in the development of an actual therapeutic method based on low-energy far-field pacing. In addition, they provide a deeper understanding of the intrinsic properties of cardiac cells.

  17. 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

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

    NASA Astrophysics Data System (ADS)

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

    1991-11-01

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

  19. Comparative Informatics Analysis to Evaluate Site-Specific Protein Oxidation in Multidimensional LC-MS/MS Data

    SciTech Connect

    McClintock, Carlee; Parks, Jerry M; Bern, Marshall; Ghattyvenkatakrishna, Pavan K; Hettich, Robert {Bob} L

    2013-01-01

    Redox proteomics has yielded molecular insight into diseases of protein dysfunction attributable to oxidative stress, underscoring the need for robust detection of protein oxidation products. Additionally, oxidative protein surface mapping techniques utilize hydroxyl radicals to gain structural insight about solvent exposure. Interpretation of tandem mass spectral data is a critical challenge for such investigations, because reactive oxygen species target a wide breadth of amino acids. Additionally, oxidized peptides may be generated in a wide range of abundances since the reactivity of hydroxyl radicals with different amino acids spans three orders of magnitude. Taken together, these attributes of oxidative footprinting pose both experimental and computational challenges to detecting oxidized peptides that are naturally less abundant than their unoxidized counterparts. In this study, three model proteins were oxidized electrochemically and analyzed at both the intact protein and peptide levels. A multidimensional chromatographic strategy was utilized to expand the dynamic range of oxidized peptides measurements. Peptide mass spectral data were searched by the hybrid software packages Inspect and Byonic, which incorporate de novo elements of spectral interpretation into a database search. This dynamic search capacity accommodates the challenge of searching for more than forty oxidative mass shifts that can occur in a staggering variety of possible combinatorial occurrences. A prevailing set of oxidized residues was identified with this comparative approach, and evaluation of these sites was informed by solvent accessible surface area gleaned through molecular dynamics simulations. Along with increased levels of oxidation around highly reactive hotspot sites as expected, the enhanced sensitivity of these measurements uncovered a surprising level of oxidation on less reactive residues.

  20. Comparative Informatics Analysis to Evaluate Site-Specific Protein Oxidation in Multidimensional LC-MS/MS Data

    PubMed Central

    McClintock, Carlee S.; Parks, Jerry M.; Bern, Marshall; GhattyVenkataKrishna, Pavan K.; Hettich, Robert L.

    2013-01-01

    Redox proteomics has yielded molecular insight into diseases of protein dysfunction attributable to oxidative stress, underscoring the need for robust detection of protein oxidation products. Additionally, oxidative protein surface mapping techniques utilize hydroxyl radicals to gain structural insight about solvent exposure. Interpretation of tandem mass spectral data is a critical challenge for such investigations, because reactive oxygen species target a wide breadth of amino acids. Additionally, oxidized peptides may be generated in a wide range of abundances since the reactivity of hydroxyl radicals with different amino acids spans three orders of magnitude. Taken together, these attributes of oxidative footprinting pose both experimental and computational challenges to detecting oxidized peptides that are naturally less abundant than their unoxidized counterparts. In this study, three model proteins were oxidized electrochemically and analyzed at both the intact protein and peptide levels. A multidimensional chromatographic strategy was utilized to expand the dynamic range of oxidized peptide measurements. Peptide mass spectral data were searched by the “hybrid” software packages Inspect and Byonic, which incorporate de novo elements of spectral interpretation into a database search. This dynamic search capacity accommodates the challenge of searching for more than forty oxidative mass shifts that can occur in a staggering variety of possible combinatorial occurrences. A prevailing set of oxidized residues was identified with this comparative approach, and evaluation of these sites was informed by solvent accessible surface area gleaned through molecular dynamics simulations. Along with increased levels of oxidation around highly reactive “hotspot” sites as expected, the enhanced sensitivity of these measurements uncovered a surprising level of oxidation on less reactive residues. PMID:23827042

  1. Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

    PubMed Central

    Zorrilla de San Martin, Javier; Jalil, Abdelali

    2015-01-01

    Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABAARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABAA autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca2+ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl−]i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABAA channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Nav-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABAA autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity. PMID:26621773

  2. Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity.

    PubMed

    de San Martin, Javier Zorrilla; Jalil, Abdelali; Trigo, Federico F

    2015-12-01

    Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABA(A)Rs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABA(A) autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca(2+) photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl(-)](i), autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30-150 GABA(A) channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Na(v)-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABA(A) autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity.

  3. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    PubMed Central

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda

    2017-01-01

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo. PMID:28045057

  4. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    NASA Astrophysics Data System (ADS)

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda

    2017-01-01

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

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

    SciTech Connect

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

    2014-10-02

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

  6. Active-site modifications of adenylation domains lead to hydrolysis of upstream nonribosomal peptidyl thioester intermediates.

    PubMed

    Uguru, Gabriel C; Milne, Claire; Borg, Matthew; Flett, Fiona; Smith, Colin P; Micklefield, Jason

    2004-04-28

    Site-directed mutagenesis of nonribosomal peptide synthetase (NRPS) adenylation (A) domains was investigated as a means to engineer new calcium-dependent antibiotics (CDA) in Streptomyces coelicolor. Single- and double-point mutants of the CDA NRPS module 7, A-domain were generated, which were predicted to alter the specificity of this domain from Asp to Asn. The double-point mutant produced a new peptide CDA2a-7N containing Asn at position 7 as expected. However, in both the single- and the double-point mutants, significant hydrolysis of the CDA-6mer intermediate was evident. One explanation for this is that the mutant module 7 A-domain activates Asn instead of Asp; however, the Asn-thioester intermediate is only weakly recognized by the upstream C-domain acceptor site (a), allowing a water molecule to intercept the hexapeptidyl intermediate in the donor site (d).

  7. Novel active comb-shaped dry electrode for EEG measurement in hairy site.

    PubMed

    Huang, Yan-Jun; Wu, Chung-Yu; Wong, Alice May-Kuen; Lin, Bor-Shyh

    2015-01-01

    Electroencephalography (EEG) is an important biopotential, and has been widely applied in clinical applications. The conventional EEG electrode with conductive gels is usually used for measuring EEG. However, the use of conductive gel also encounters with the issue of drying and hardening. Recently, many dry EEG electrodes based on different conductive materials and techniques were proposed to solve the previous issue. However, measuring EEG in the hairy site is still a difficult challenge. In this study, a novel active comb-shaped dry electrode was proposed to measure EEG in hairy site. Different form other comb-shaped or spike-shaped dry electrodes, it can provide more excellent performance of avoiding the signal attenuation, phase distortion, and the reduction of common mode rejection ratio. Even under walking motion, it can effectively acquire EEG in hairy site. Finally, the experiments for alpha rhythm and steady-state visually evoked potential were also tested to validate the proposed electrode.

  8. Substrate conformational transitions in the active site of chorismate mutase: their role in the catalytic mechanism.

    PubMed

    Guo, H; Cui, Q; Lipscomb, W N; Karplus, M

    2001-07-31

    Chorismate mutase acts at the first branch-point of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. The results of molecular dynamics simulations of the substrate in solution and in the active site of chorismate mutase are reported. Two nonreactive conformers of chorismate are found to be more stable than the reactive pseudodiaxial chair conformer in solution. It is shown by QM/MM molecular dynamics simulations, which take into account the motions of the enzyme, that when these inactive conformers are bound to the active site, they are rapidly converted to the reactive chair conformer. This result suggests that one contribution of the enzyme is to bind the more prevalent nonreactive conformers and transform them into the active form in a step before the chemical reaction. The motion of the reactive chair conformer in the active site calculated by using the QM/MM potential generates transient structures that are closer to the transition state than is the stable CHAIR conformer.

  9. How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism

    PubMed Central

    Hytönen, Vesa P; Vogel, Viola

    2008-01-01

    Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds. PMID:18282082

  10. Progress report on decommissioning activities at the Fernald Environmental Management Project (FEMP) site

    SciTech Connect

    1998-07-01

    The Fernald Environmental Management Project (FEMP), is located about 18 miles northwest of Cincinnati, Ohio. Between 1953 and 1989, the facility, then called the Feed Material Production Center or FMPC, produced uranium metal products used in the eventual production of weapons grade material for use by other US Department of Energy (DOE) sites. In 1989, FMPC`s production was suspended by the federal government in order to focus resources on environmental restoration versus defense production. In 1992, Fluor Daniel Fernald assumed responsibility for managing all cleanup activities at the FEMP under contract to the DOE. In 1990, as part of the remediation effort, the site was divided into five operable units based on physical proximity of contaminated areas, similar amounts of types of contamination, or the potential for a similar technology to be used in cleanup activities. This report continues the outline of the decontamination and decommissioning (D and D) activities at the FEMP site Operable Unit 3 (OU3) and provides an update on the status of the decommissioning activities. OU3, the Facilities Closure and Demolition Project, involves the remediation of more than 200 uranium processing facilities. The mission of the project is to remove nuclear materials stored in these buildings, then perform the clean out of the buildings and equipment, and decontaminate and dismantle the facilities.

  11. Structure-based drug design: exploring the proper filling of apolar pockets at enzyme active sites.

    PubMed

    Zürcher, Martina; Diederich, François

    2008-06-20

    The proper filling of apolar pockets at enzyme active sites is central for increasing binding activity and selectivity of hits and leads in medicinal chemistry. In our structure-based design approach toward the generation of potent enzyme inhibitors, we encountered a variety of challenges in gaining suitable binding affinity from the occupation of such pockets. We summarize them here for the first time. A fluorine scan of tricyclic thrombin inhibitors led to the discovery of favorable orthogonal dipolar C-F...CO interactions. Efficient cation-pi interactions were established in the S4 pocket of factor Xa, another serine protease from the blood coagulation cascade. Changing from mono- to bisubstrate inhibitors of catechol O-methyltransferase, a target in the L-Dopa-based treatment of Parkinson's disease, enabled the full exploitation of a previously unexplored hydrophobic pocket. Conformational preorganization of a pocket at an enzyme active site is crucial for harvesting binding affinity. This is demonstrated for two enzymes from the nonmevalonate pathway of isoprenoid biosynthesis, IspE and IspF, which are pursued as antimalarial targets. Disrupting crystallographically defined water networks on the way into a pocket might cost all of the binding free enthalpy gained from its occupation, as revealed in studies with tRNA-guanine transglycosylase, a target against shigellosis. Investigations of the active site of plasmepsin II, another antimalarial target, showed that principles for proper apolar cavity filling, originally developed for synthetic host-guest systems, are also applicable to enzyme environments.

  12. A NMR and MD study of the active site of factor Xa by selective inhibitors

    NASA Astrophysics Data System (ADS)

    Doan, B. T.; Fraternali, F.; Do, Q. T.; Atkinson, R. A.; Palmas, P.; Sklenar, V.; Wildgoose, P.; Strop, P.; Saudek, V.

    1998-02-01

    The structure of two selective inhibitors obtained by the screening of a vast combinatorial library, Ac-Tyr-Ile-Arg-Ile-NH2 and Ac-(4-amino-Phe)-(Cyc.-Gly)-NH2, in the active site of the blood clotting enzyme factor Xa was determined using transferred NOE NMR and simulated annealing (SA) under NMR constraints. The refined structures of the inhibitors were docked in the active site and SA was performed inside the enzyme which has been kept as a rigid charged template. The final structures were optimised by molecular dynamics simulation of the complexes in water. The inhibitors assume a compact, very well defined conformation embedded in the binding site without blocking the catalysis. The model allows to explain the mode of action, affinity and specificity. L'étude structurale d'inhibiteurs du facteur Xa, une enzyme de coagulation, obtenus par chimie combinatoire : Ac-Tyr-Ile-Arg-Ile-NH2, Ac-(4-amino-Phe)-(Cyc.-Gly)-NH2, a été réalisée par RMN NOE de transfert et modélisation moléculaire. Les structures ont été calculées sous contraintes RMN : géométrie de distance, recuit simulé et minimisation, affinées par une recherche conformationnelle et recuit de l'inhibiteur placé dans le site actif et optimisées par simulation de dynamique moléculaire du complexe dans l'eau. L'inhibiteur présente une structure compacte positionnée dans le site d'interaction hors d'accès du site catalytique. Ce modèle permet d'expliquer le mode d'action, l'affinité et la spécificité des peptides.

  13. Crystal Structures and Functional Characterization of Wild Type and Active Sites Mutants of CYP101D1

    PubMed Central

    Batabyal, Dipanwita; Poulos, Thomas L.

    2014-01-01

    Although CYP101D1 and P450cam catayze the same reaction at a similar rate and share strikingly similar active site architectures, there are significance functional differences. CYP101D1 thus provides an opportunity to probe what structural and functional features must be shared and what can differ yet maintain high catalytic efficiency. Crystal structures of the cyanide complex of wild type CYP101D1 and it active site mutants, D259N and T260A, have been solved. The conformational changes in CYP101D1 upon cyanide binding are very similar to P450cam indicating a similar mechanism for proton delivery during oxygen activation using solvent assisted proton transfer. The D259N-CN− complex shows a perturbed solvent structure compared to wild type which is similar to what was observed in the oxy-complex of the corresonding D251N mutant in P450cam. As in P450cam the T260A mutant is highly uncoupled while the D259N gives barely detectable activity. Despite these similarities, CYP101D1 is able to use the P450cam redox partners while P450cam cannot use the CYP101D1 redox partners. Thus the strict requirement of P450cam for its own redox partner is relaxed in CYP101D1. Differences in the local environment of the essential Asp (Asp259 in CYP101D1) provides a strucutral basis for understanding these functional differences. PMID:24261604

  14. A unique geometry of the active site of angiotensin-converting enzyme consistent with structure-activity studies

    NASA Astrophysics Data System (ADS)

    Mayer, Dorica; Naylor, Christopher B.; Motoc, Ioan; Marshall, Garland R.

    1987-04-01

    Previous structure-activity studies of captopril and related active angiotensin-converting enzyme (ACE) inhibitors have led to the conclusion that the basic structural requirements for inhibition of ACE involve (a) a terminal carboxyl group; (b) an amido carbonyl group; and (c) different types of effective zinc (Zn) ligand functional groups. Such structural requirements common to a set of compounds acting at the same receptor have been used to define a pharmacophoric pattern of atoms or groups of atoms mutually oriented in space that is necessary for ACE inhibition from a stereochemical point of view. A unique pharmacophore model (within the resolution of approximately 0.15 Å) was observed using a method for systematic search of the conformational hyperspace available to the 28 structurally different molecules under study. The method does not assume a common molecular framework, and, therefore, allows comparison of different compounds that is independent of their absolute orientation. Consequently, by placing the carboxyl binding group, the binding site for amido carbonyl, and the Zn atom site in positions determined by ideal binding geometry with the inhibitors' functional groups, it was possible to clearly specify a geometry for the active site of ACE.

  15. Structure of a Clostridium botulinum C143S thiaminase I/thiamin complex reveals active site architecture .

    PubMed

    Sikowitz, Megan D; Shome, Brateen; Zhang, Yang; Begley, Tadhg P; Ealick, Steven E

    2013-11-05

    Thiaminases are responsible for the degradation of thiamin and its metabolites. Two classes of thiaminases have been identified based on their three-dimensional structures and their requirements for a nucleophilic second substrate. Although the reactions of several thiaminases have been characterized, the physiological role of thiamin degradation is not fully understood. We have determined the three-dimensional X-ray structure of an inactive C143S mutant of Clostridium botulinum (Cb) thiaminase I with bound thiamin at 2.2 Å resolution. The C143S/thiamin complex provides atomic level details of the orientation of thiamin upon binding to Cb-thiaminase I and the identity of active site residues involved in substrate binding and catalysis. The specific roles of active site residues were probed by using site directed mutagenesis and kinetic analyses, leading to a detailed mechanism for Cb-thiaminase I. The structure of Cb-thiaminase I is also compared to the functionally similar but structurally distinct thiaminase II.

  16. Activity of Cu{sup 2+} ions on the tetrahedral and octahedral sites of spinel oxide catalysts for CO oxidation

    SciTech Connect

    Ghose, J.; Murthy, K.S.R.C.

    1996-09-01

    In studies of CO oxidation on substituted copper chromite spinel oxide catalyst decreases as the Cu{sup 2+} content of the catalyst decreases, either by substitution with a divalent ion, i.e., Cu{sub 1-x} Mg{sub x} [Cr{sub 2}]O{sub 4}, or by reduction of Cu{sup 2+} to Cu{sup 1+}. Crystallographic studies have shown that Cu[Cr{sub 2}]O{sub 4} changes from normal to partially inverse when Cr{sup 3+} is replaced by Al{sup 3+}. Thus, in aluminum-substituted copper chromite catalysts, copper is present on both tetrahedral and octahedral sites of the spinel lattice, i.e., Cu{sub 1-x}Al{sub x} [Cu{sub x}Cr{sub 2-(x+y)}Al{sub y}]O{sub 4}. ESCA studies have shown that upon Al substitution some of the tetrahedral Cu{sup 2+} ions are reduced to Cu{sup 1+} and this causes a reduction in the catalytic activity of the catalysts. The present work was taken up to compare the activity of Cu{sup 2+} on tetrahedral sites with that on octahedral sites of the spinel oxide catalysts. For this, CO oxidation studies were carried out on the inverse spinel CuFe{sub 2}O{sub 4} and on the normal spinel CuRh{sub 2}O{sub 4} catalysts. 7 refs., 1 fig.

  17. Identification of a novel K311 ubiquitination site critical for androgen receptor transcriptional activity.

    PubMed

    McClurg, Urszula L; Cork, David M W; Darby, Steven; Ryan-Munden, Claudia A; Nakjang, Sirintra; Mendes Côrtes, Leticia; Treumann, Achim; Gaughan, Luke; Robson, Craig N

    2016-11-29

    The androgen receptor (AR) is the main driver of prostate cancer (PC) development and progression, and the primary therapeutic target in PC. To date, two functional ubiquitination sites have been identified on AR, both located in its C-terminal ligand binding domain (LBD). Recent reports highlight the emergence of AR splice variants lacking the LBD that can arise during disease progression and contribute to castrate resistance. Here, we report a novel N-terminal ubiquitination site at lysine 311. Ubiquitination of this site plays a role in AR stability and is critical for its transcriptional activity. Inactivation of this site causes AR to accumulate on chromatin and inactivates its transcriptional function as a consequence of inability to bind to p300. Additionally, mutation at lysine 311 affects cellular transcriptome altering the expression of genes involved in chromatin organization, signaling, adhesion, motility, development and metabolism. Even though this site is present in clinically relevant AR-variants it can only be ubiquitinated in cells when AR retains LBD suggesting a role for AR C-terminus in E2/E3 substrate recognition. We report that as a consequence AR variants lacking the LBD cannot be ubiquitinated in the cellular environment and their protein turnover must be regulated via an alternate pathway.

  18. Foreign Glycoproteins Can Be Actively Recruited to Virus Assembly Sites during Pseudotyping▿

    PubMed Central

    Jorgenson, Rebecca L.; Vogt, Volker M.; Johnson, Marc C.

    2009-01-01

    Retroviruses like human immunodeficiency virus type 1 (HIV-1), as well as many other enveloped viruses, can efficiently produce infectious virus in the absence of their own surface glycoprotein if a suitable glycoprotein from a foreign virus is expressed in the same cell. This process of complementation, known as pseudotyping, often can occur even when the glycoprotein is from an unrelated virus. Although pseudotyping is widely used for engineering chimeric viruses, it has remained unknown whether a virus can actively recruit foreign glycoproteins to budding sites or, alternatively, if a virus obtains the glycoproteins through a passive mechanism. We have studied the specificity of glycoprotein recruitment by immunogold labeling viral glycoproteins and imaging their distribution on the host plasma membrane using scanning electron microscopy. Expressed alone, all tested viral glycoproteins were relatively randomly distributed on the plasma membrane. However, in the presence of budding HIV-1 or Rous sarcoma virus (RSV) particles, some glycoproteins, such as those encoded by murine leukemia virus and vesicular stomatitis virus, were dramatically redistributed to viral budding sites. In contrast, the RSV Env glycoprotein was robustly recruited only to the homologous RSV budding sites. These data demonstrate that viral glycoproteins are not in preformed membrane patches prior to viral assembly but rather that glycoproteins are actively recruited to certain viral assembly sites. PMID:19224995

  19. Barriers to physical activity in an on-site corporate fitness center.

    PubMed

    Schwetschenau, Heather M; O'Brien, William H; Cunningham, Christopher J L; Jex, Steve M

    2008-10-01

    Many corporations provide employees the option of participating in on-site fitness centers, but utilization rates are low. Perceived barriers to physical activity have been established as important correlates of physical activity, and recent research indicates that barriers may vary across settings. Work-site fitness centers may present unique barriers to participation, but there are currently no standardized measures that assess such barriers. Eighty-eight employees of a midwestern corporation completed a survey designed to identify and evaluate the extent to which barriers influence participation in an on-site corporate fitness center. Regression analyses revealed that external environmental barriers (e.g., inadequate exercise facilities) significantly accounted for not joining the fitness center, and for decreased duration of visits to the facility among members. Internal barriers (e.g., feeling embarrassed to exercise around coworkers) significantly accounted for frequency of fitness center visits among members. This corporate specific measure may lead to more effective interventions aimed to increase use of on-site corporate fitness centers.

  20. Den site activity patterns of adult male and female swift foxes, Vulpes velox, in Northwestern Texas

    USGS Publications Warehouse

    Lemons, P.R.; Ballard, W.B.; Sullivan, R.M.; Sovada, M.A.

    2003-01-01

    Activity of Swift Foxes (Vulpes velox) at den sites was studied in northwestern Texas during pup rearing seasons in 2000 and 2001 to determine role of males in parental care. Twenty-four percent of radio-collared females with a potential to breed successfully raised pups to eight weeks of age. We intensively monitored presence and absence of male and female Swift Foxes at two den sites each year. Females were present >2.6 times more at den sites than males during the pup rearing season. Female and male Swift Foxes largely stayed at dens during diurnal hours and were active away from dens during nocturnal and crepuscular hours. Females and males spent 12.4% and 3.0% more time at dens before pups emerged, than after pups emerged, respectively. Following depredation of one male parent, the female spent 29% less time at the den site. Decrease in time spent at the den by the female following loss of her mate suggested that loss of one parent might severely impact recruitment of Swift Foxes. Our observations indicated that intense Coyote (Canis latrans) depredation may severely impact pup-rearing success as well as the parental care within Swift Fox family groups.

  1. 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

  2. Linde FUSRAP Site Remediation: Engineering Challenges and Solutions of Remedial Activities on an Active Industrial Facility - 13506

    SciTech Connect

    Beres, Christopher M.; Fort, E. Joseph; Boyle, James D.

    2013-07-01

    The Linde FUSRAP Site (Linde) is located in Tonawanda, New York at a major research and development facility for Praxair, Inc. (Praxair). Successful remediation activities at Linde combines meeting cleanup objectives of radiological contamination while minimizing impacts to Praxair business operations. The unique use of Praxair's property coupled with an array of active and abandoned utilities poses many engineering and operational challenges; each of which has been overcome during the remedial action at Linde. The U.S. Army Corps of Engineers - Buffalo District (USACE) and CABRERA SERVICES, INC. (CABRERA) have successfully faced engineering challenges such as relocation of an aboveground structure, structural protection of an active water line, and installation of active mechanical, electrical, and communication utilities to perform remediation. As remediation nears completion, continued success of engineering challenges is critical as remaining activities exist in the vicinity of infrastructure essential to business operations; an electrical substation and duct bank providing power throughout the Praxair facility. Emphasis on engineering and operations through final remediation and into site restoration will allow for the safe and successful completion of the project. (authors)

  3. Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements of the amount of lipase immobilized (bicinchoninic acid method) and the catalyst’s tributyrin hydrolysis activity, coupled with a determination of the lipase’s functional fr...

  4. Mutation of active site residues in synthetic T4-lysozyme gene and their effect on lytic activity.

    PubMed

    Anand, N N; Stephen, E R; Narang, S A

    1988-06-16

    The active site amino acids (Glu11 and Asp20) in T4-lysozyme have been mutated to their isosteric residues Gln or Asn and/or acidic residues such as Glu----Asp or Asp----Glu by the oligonucleotide-replacement method. Out of eight mutants so generated the mutant T4-lysozyme obtained from pTLY.Asp11 retains maximum amount of activity (approximately 16%), pTLY.Asn20 the least (0.9%) whereas pTLY.Gln11 lost completely. A systematic study of the active and inactive mutants thus generated supports the important role of Glu11 and Asp20 in T4-lysozyme activity as predicted in earlier studies.

  5. 78 FR 21352 - Update on Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-10

    ... on Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY... reimbursement for cleanup work performed by licensees at eligible uranium and thorium processing sites in... licensees of eligible uranium and thorium processing sites. If licensees submit claims in FY 2013,...

  6. Synthesis of supported bimetallic nanoparticles with controlled size and composition distributions for active site elucidation

    SciTech Connect

    Hakim, Sikander H.; Sener, Canan; Alba Rubio, Ana C.; Gostanian, Thomas M.; O'neill, Brandon J; Ribeiro, Fabio H.; Miller, Jeffrey T.; Dumesic, James A

    2015-08-01

    Elucidation of active sites in supported bimetallic catalysts is complicated by the high level of dispersity in the nanoparticle size and composition that is inherent in conventional methods of catalyst preparation. We present a synthesis strategy that leads to highly dispersed, bimetallic nanoparticles with uniform particle size and composition by means of controlled surface reactions. We demonstrate the synthesis of three systems, RhMo, PtMo, and RhRe, consisting of a highly reducible metal with an oxophilic promoter. These catalysts are characterized by FTIR, CO chemisorption, STEM/EDS, TPR, and XAS analysis. The catalytic properties of these bimetallic nanoparticles were probed for the selective CO hydrogenolysis of (hydroxymethyl)tetrahydropyran to produce 1,6 hexanediol. Based on the characterization results and reactivity trends, the active sites in the hydrogenolysis reaction are identified to be small ensembles of the more noble metal (Rh, Pt) adjacent to highly reduced moieties of the more oxophilic metal (Mo, Re).

  7. Dynamics and Mechanism of Efficient DNA Repair Reviewed by Active-Site Mutants

    NASA Astrophysics Data System (ADS)

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

    2010-06-01

    Photolyases repair the UV-induced pyrimidine dimers in damage DNA via a photoreaction which includes a series of light-driven electron transfers between the two-electron-reduced flavin cofactor FADH^- and the dimer. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of several active-site residues. With femtosecond resolution, we observed the significant change in the forward electron transfer from the excited FADH^- to the dimer and the back electron transfer from the repaired thymines by mutation of E274A, R226A, R342A, N378S and N378C. We also found that the mutation of E274A accelerates the bond-breaking of the thymine dimer. The dynamics changes are consistent with the quantum yield study of these mutants. These results suggest that the active-site residues play a significant role, structurally and chemically, in the DNA repair photocycle.

  8. Extreme electric fields power catalysis in the active site of ketosteroid isomerase.

    PubMed

    Fried, Stephen D; Bagchi, Sayan; Boxer, Steven G

    2014-12-19

    Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these electric fields have proven difficult to quantify with standard experimental approaches. Using vibrational Stark effect spectroscopy, we found that the active site of the enzyme ketosteroid isomerase (KSI) exerts an extremely large electric field onto the C=O chemical bond that undergoes a charge rearrangement in KSI's rate-determining step. Moreover, we found that the magnitude of the electric field exerted by the active site strongly correlates with the enzyme's catalytic rate enhancement, enabling us to quantify the fraction of the catalytic effect that is electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomolecular systems.

  9. The active site of RNA polymerase II participates in transcript cleavage within arrested ternary complexes.

    PubMed Central

    Rudd, M D; Izban, M G; Luse, D S

    1994-01-01

    RNA polymerase II may become arrested during transcript elongation, in which case the ternary complex remains intact but further RNA synthesis is blocked. To relieve arrest, the nascent transcript must be cleaved from the 3' end. RNAs of 7-17 nt are liberated and transcription continues from the newly exposed 3' end. Factor SII increases elongation efficiency by strongly stimulating the transcript cleavage reaction. We show here that arrest relief can also occur by the addition of pyrophosphate. This generates the same set of cleavage products as factor SII, but the fragments produced with pyrophosphate have 5'-triphosphate termini. Thus, the active site of RNA polymerase II, in the presence of pyrophosphate, appears to be capable of cleaving phosphodiester linkages as far as 17 nt upstream of the original site of polymerization, leaving the ternary complex intact and transcriptionally active. Images PMID:8058756

  10. Twinning in fcc lattice creates low-coordinated catalytically active sites in porous gold

    NASA Astrophysics Data System (ADS)

    Krajčí, Marian; Kameoka, Satoshi; Tsai, An-Pang

    2016-08-01

    We describe a new mechanism for creation of catalytically active sites in porous gold. Samples of porous gold prepared by de-alloying Al2Au exhibit a clear correlation between the catalytic reactivity towards CO oxidation and structural defects in the fcc lattice of Au. We have found that on the stepped {211} surfaces quite common twin boundary defects in the bulk structure of porous gold can form long close-packed rows of atoms with the coordination number CN = 6. DFT calculations confirm that on these low-coordinated Au sites dioxygen chemisorbs and CO oxidation can proceed via the Langmuir-Hinshelwood mechanism with the activation energy of 37 kJ/mol or via the CO-OO intermediate with the energy barrier of 19 kJ/mol. The existence of the twins in porous gold is stabilized by the surface energy.

  11. Twinning in fcc lattice creates low-coordinated catalytically active sites in porous gold.

    PubMed

    Krajčí, Marian; Kameoka, Satoshi; Tsai, An-Pang

    2016-08-28

    We describe a new mechanism for creation of catalytically active sites in porous gold. Samples of porous gold prepared by de-alloying Al2Au exhibit a clear correlation between the catalytic reactivity towards CO oxidation and structural defects in the fcc lattice of Au. We have found that on the stepped {211} surfaces quite common twin boundary defects in the bulk structure of porous gold can form long close-packed rows of atoms with the coordination number CN = 6. DFT calculations confirm that on these low-coordinated Au sites dioxygen chemisorbs and CO oxidation can proceed via the Langmuir-Hinshelwood mechanism with the activation energy of 37 kJ/mol or via the CO-OO intermediate with the energy barrier of 19 kJ/mol. The existence of the twins in porous gold is stabilized by the surface energy.

  12. 13C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites

    PubMed Central

    McCullough, Christopher R.; Pullela, Phani Kumar; Im, Sang-Choul; Waskell, Lucy

    2012-01-01

    The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a 13CH3-reporter attached. This 13C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site. PMID:19199046

  13. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    PubMed

    Wang, P-H; De Sancho, D; Best, R B; Blumberger, J

    2016-01-01

    The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates.

  14. Neurovestibular toxicities of acrylonitrile and iminodipropionitrile in rats: a comparative evaluation of putative mechanisms and target sites.

    PubMed

    Khan, Haseeb Ahmad; Alhomida, Abdullah Saleh; Arif, Ibrahim Abdulwahid

    2009-05-01

    This investigation was aimed to study the effects of individual and concomitant exposures of the two nitrile compounds, the industrially important acrylonitrile (ACN; 5, 15, 45 mg/kg/day) and the positive control iminodipropionitrile (IDPN; 100 mg/kg/day) in rats. The six treatment groups were 1 (control), 2 (ACN 5), 3 (ACN 15), 4 (ACN 45), 5 (IDPN), and 6 (IDPN + ACN 15). Both the drugs were started on the same day and continued for 9 days (IDPN was given daily 30 min before ACN but stopped a day earlier). The animals were daily observed for neurobehavioral abnormalities including dyskinetic head movements, circling, tail hanging, air righting reflex, and contact inhibition of righting reflex. There was no dyskinetic behavioral abnormality in the animals treated with any of the three doses of ACN whereas all the rats in IDPN alone treated group developed clear symptoms of excitation, circling, and chorea syndrome (ECC syndrome) on day 9. Concomitant treatment of rats with ACN significantly attenuated the severity of IDPN-induced behavioral deficits. Administration of ACN significantly depleted glutathione (GSH) in striatum, hippocampus and cerebral cortex; IDPN significantly reduced the GSH only in striatum. The anterior striatum showed intense tyrosine hydroxylase (TH) expression in IDPN alone treated rat as compared to control and ACN alone treated rat. Cotreatment with ACN reduced the intensity of TH immunostaining in IDPN-treated rats. Administration of IDPN alone caused massive loss of vestibular sensory hair cells in the crista ampullaris whereas the sensory epithelium appeared intact in ACN alone treated groups. The animals receiving the combination of ACN and IDPN showed comparatively less degeneration of sensory hair cells than IDPN alone group. These findings suggest that ACN and IDPN produce different behavioral effects that are exerted through entirely different mechanisms; the nervous and vestibular systems appear to be the major target sites of these

  15. Improved Biofilm Antimicrobial Activity of Polyethylene Glycol Conjugated Tobramycin Compared to Tobramycin in Pseudomonas aeruginosa Biofilms.

    PubMed

    Du, Ju; Bandara, H M H N; Du, Ping; Huang, Hui; Hoang, Khang; Nguyen, Dang; Mogarala, Sri Vasudha; Smyth, Hugh D C

    2015-05-04

    The objective of this study was to develop a functionally enhanced antibiotic that would improve the therapeutic activity against bacterial biofilms. Tobramycin was chemically conjugated with polyethylene glycol (PEG) via site-specific conjugation to form PEGylated-tobramycin (Tob-PEG). The antibacterial efficacy of Tob-PEG, as compared to tobramycin, was assessed on the planktonic phase and biofilms phase of Pseudomonas aeruginosa. The minimum inhibitory concentration (MIC80) of Tob-PEG was higher (13.9 μmol/L) than that of tobramycin (1.4 μmol/L) in the planktonic phases. In contrast, the Tob-PEG was approximately 3.2-fold more effective in eliminating bacterial biofilms than tobramycin. Specifically, Tob-PEG had a MIC80 lower than those exhibited by tobramycin (27.8 μmol/L vs 89.8 μmol/L). Both confocal laser scanning microscopy and scanning electron microscopy further confirmed these data. Thus, modification of antimicrobials by PEGylation appears to be a promising approach for overcoming the bacterial resistance in the established biofilms of Pseudomonas aeruginosa.

  16. Standard and generalized McDonald-Kreitman test: a website to detect selection by comparing different classes of DNA sites.

    PubMed

    Egea, Raquel; Casillas, Sònia; Barbadilla, Antonio

    2008-07-01

    The McDonald and Kreitman test (MKT) is one of the most powerful and extensively used tests to detect the signature of natural selection at the molecular level. Here, we present the standard and generalized MKT website, a novel website that allows performing MKTs not only for synonymous and nonsynonymous changes, as the test was initially described, but also for other classes of regions and/or several loci. The website has three different interfaces: (i) the standard MKT, where users can analyze several types of sites in a coding region, (ii) the advanced MKT, where users can compare two closely linked regions in the genome that can be either coding or noncoding, and (iii) the multi-locus MKT, where users can analyze many separate loci in a single multi-locus test. The website has already been used to show that selection efficiency is positively correlated with effective population size in the Drosophila genus and it has been applied to include estimates of selection in DPDB. This website is a timely resource, which will presumably be widely used by researchers in the field and will contribute to enlarge the catalogue of cases of adaptive evolution. It is available at http://mkt.uab.es.

  17. [Comparative evaluation of the toxicity induced by mussels (Mytilus galloprovincialis) from two sites of the Moroccan Atlantic coast in mice].

    PubMed

    Moustaid, Khadija; Nasser, Boubker; Baudrimont, Isabelle; Anane, Rachid; El Idrissi, Mostafa; Bouzidi, Abdelhamid; Creppy, Edmond E

    2005-03-01

    Atlantic coast in mice. Preliminary studies showed that seawater contains heavy metals from domestic, agricultural and industrial wastes. Marine bivalves concentrate these pollutants by filtration and serve as vectors in human exposure. The objective of this study was to determine the concentration of heavy metals; cadmium (Cd); chromium (Cr), and lead (Pb) in mussels (Mytilus galloprovincialis) collected in two coastal sites; Jorf Lasfar (JL) (neighbouring a phosphate processing platform) and Oualidia (OL) (a vegetable growing area) located at 120 and 190 km south of Casablanca, respectively. Another objective was to test and compare the toxicity of these mussels on mice. The results indicated the presence of heavy metals (Cd, Cr, and Pb) in mussels at different concentrations, depending on the collection period. Higher concentrations were obtained at JL than at OL: for example, Cd concentrations were 80 +/- 15 to 199 +/- 28 versus 23 +/- 5 microg/g mussel dry weight, respectively. Cramming with mussel powder did not increase Cd, Cr, or Pb concentration in either liver or kidneys of treated mice. The relative kidney weights were reduced. Increased glucose urea was observed in animals' urine. Treatment with mussels from OL induced significant reduction (20%) in mice body weight, together with an increase in creatinuria. These results indicate that mussels collected from OL are more harmful than those obtained from JL are. All these mussels should not be recommended for human consumption.

  18. Standard and generalized McDonald–Kreitman test: a website to detect selection by comparing different classes of DNA sites

    PubMed Central

    Egea, Raquel; Casillas, Sònia; Barbadilla, Antonio

    2008-01-01

    The McDonald and Kreitman test (MKT) is one of the most powerful and extensively used tests to detect the signature of natural selection at the molecular level. Here, we present the standard and generalized MKT website, a novel website that allows performing MKTs not only for synonymous and nonsynonymous changes, as the test was initially described, but also for other classes of regions and/or several loci. The website has three different interfaces: (i) the standard MKT, where users can analyze several types of sites in a coding region, (ii) the advanced MKT, where users can compare two closely linked regions in the genome that can be either coding or noncoding, and (iii) the multi-locus MKT, where users can analyze many separate loci in a single multi-locus test. The website has already been used to show that selection efficiency is positively correlated with effective population size in the Drosophila genus and it has been applied to include estimates of selection in DPDB. This website is a timely resource, which will presumably be widely used by researchers in the field and will contribute to enlarge the catalogue of cases of adaptive evolution. It is available at http://mkt.uab.es. PMID:18515345

  19. A comparative 'bottom up' proteomics strategy for the site-specific identification and quantification of protein modifications by electrophilic lipids.

    PubMed

    Han, Bingnan; Hare, Michael; Wickramasekara, Samanthi; Fang, Yi; Maier, Claudia S

    2012-10-22

    We report a mass spectrometry-based comparative "bottom up" proteomics approach that combines d(0)/d(4)-succinic anhydride labeling with commercially available hydrazine (Hz)-functionalized beads (Affi-gel Hz beads) for detection, identification and relative quantification of site-specific oxylipid modifications in biological matrices. We evaluated and applied this robust and simple method for the quantitative analysis of oxylipid protein conjugates in cardiac mitochondrial proteome samples isolated from 3- and 24-month-old rat hearts. The use of d(0)/d(4)-succinic anhydride labeling, Hz-bead based affinity enrichment, nanoLC fractionation and MALDI-ToF/ToF tandem mass spectrometry yielded relative quantification of oxylipid conjugates with residue-specific modification information. Conjugation of acrolein (ACR), 4-hydroxy-2-hexenal (HHE), 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-noneal (ONE) to cysteine, histidine and lysine residues were identified. HHE conjugates were the predominant subset of Michael-type adducts detected in this study. The HHE conjugates showed higher levels in mitochondrial preparations from young heart congruent with previous findings by others that the n-3/n-6 PUFA ratio is higher in young heart mitochondrial membranes. Although this study focuses on protein adducts of reactive oxylipids, the method might be equally applicable to protein carbonyl modifications caused by metal catalyzed oxidation reactions.

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

    NASA Astrophysics Data System (ADS)

    Vasileiou, Eleni; Kallioras, Andreas

    2016-04-01

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

  1. BIOPHYSICS. Comment on "Extreme electric fields power catalysis in the active site of ketosteroid isomerase".

    PubMed

    Natarajan, Aditya; Yabukarski, Filip; Lamba, Vandana; Schwans, Jason P; Sunden, Fanny; Herschlag, Daniel

    2015-08-28

    Fried et al. (Reports, 19 December 2014, p. 1510) demonstrated a strong correlation between reaction rate and the carbonyl stretching frequency of a product analog bound to ketosteroid isomerase oxyanion hole mutants and concluded that the active-site electric field provides 70% of catalysis. Alternative comparisons suggest a smaller contribution, relative to the corresponding solution reaction, and highlight the importance of atomic-level descriptions.

  2. Disposal Activities and the Unique Waste Streams at the Nevada National Security Site (NNSS)

    SciTech Connect

    Arnold, P.

    2012-10-31

    This slide show documents waste disposal at the Nevada National Security Site. Topics covered include: radionuclide requirements for waste disposal; approved performance assessment (PA) for depleted uranium disposal; requirements; program approval; the Waste Acceptance Review Panel (WARP); description of the Radioactive Waste Acceptance Program (RWAP); facility evaluation; recent program accomplishments, nuclear facility safety changes; higher-activity waste stream disposal; and, large volume bulk waste streams.

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

    PubMed Central

    Sumner, Isaiah; Iyengar, Srinivasan S.

    2010-01-01

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

  4. Non-specific binding sites help to explain mixed inhibition in mushroom tyrosinase activities.

    PubMed

    Hassani, Sorour; Haghbeen, Kamahldin; Fazli, Mostafa

    2016-10-21

    Inhibition and activation studies of tyrosinase could prove beneficial to agricultural, food, cosmetic, and pharmaceutical industries. Although non-competitive and mixed-inhibition are frequent modes observed in kinetics studies on mushroom tyrosinase (MT) activities, the phenomena are left unexplained. In this study, dual effects of phthalic acid (PA) and cinnamic acid (CA) on MT during mono-phenolase activity were demonstrated. PA activated and inhibited MT at concentrations lower and higher than 150 μM, respectively. In contrast, CA inhibited and activated MT at concentrations lower and higher than 5 μM. The mode of inhibition for both effectors was mixed-type. Complex kinetics of MT in the presence of a modulator could partly be ascribed to its mixed-cooperativity. However, to explain mixed-inhibition mode, it is necessary to demonstrate how the ternary complex of substrate/enzyme/effector is formed. Therefore, we looked for possible non-specific binding sites using MT tropolone-bound PDB (2Y9X) in the computational studies. When tropolone was in MTPa (active site), PA and CA occupied different pockets (named MTPb and MTPc, respectively). The close Moldock scores of PA binding posed in MTPb and MTPa suggested that MTPb could be a secondary binding site for PA. Similar results were obtained for CA. Ensuing results from 10 ns molecular dynamics simulations for 2Y9X-effector complexes indicated that the structures were gradually stabilized during simulation. Tunnel analysis by using CAVER Analyst and CHEXVIS resulted in identifying two distinct channels that assumingly participate in exchanging the effectors when the direct channel to MTPa is not accessible.

  5. Crystal Structure of Albaflavenone Monooxygenase Containing a Moonlighting Terpene Synthase Active Site*

    PubMed Central

    Zhao, Bin; Lei, Li; Vassylyev, Dmitry G.; Lin, Xin; Cane, David E.; Kelly, Steven L.; Yuan, Hang; Lamb, David C.; Waterman, Michael R.

    2009-01-01

    Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 Å) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 Å). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 α-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an α-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein. PMID:19858213

  6. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    SciTech Connect

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J.

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  7. Site-specific mutagenesis and functional analysis of active sites of sulfur oxygenase reductase from Gram-positive moderate thermophile Sulfobacillus acidophilus TPY.

    PubMed

    Zhang, Huijun; Guo, Wenbin; Xu, Changan; Zhou, Hongbo; Chen, Xinhua

    2013-12-14

    Sequence alignments revealed that the conserved motifs of SORSa which formed an independent branch between archaea and Gram-negative bacteria SORs according to the phylogenetic relationship were similar with the archaea and Gram-negative bacteria SORs. In order to investigate the active sites of SORSa, cysteines 31, 101 and 104 (C31, C101, C104), histidines 86 and 90 (H86 and H90) and glutamate 114 (E114) of SORSa were chosen as the target amino acid residues for site-specific mutagenesis. The wild type and six mutant SORs were expressed in E. coli BL21, purified and confirmed by SDS-PAGE and Western blotting analysis. Enzyme activity determination revealed that the active sites of SORSa were identical with the archaea and Gram-negative bacteria SORs reported. Replacement of any cysteine residues reduced SOR activity by 53-100%, while the mutants of H86A, H90A and E114A lost their enzyme activities largely, only remaining 20%, 19% and 32% activity of the wild type SOR respectively. This study will enrich our awareness for active sites of SOR in a Gram-positive bacterium.

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

    PubMed Central

    Herschlag, Daniel

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-01

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5‧-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

  10. A Relaxed Active Site After Exon Ligation by the Group I Intron

    SciTech Connect

    Lipchock,S.; Strobel, S.

    2008-01-01

    During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. Here, we report the crystal structure of the intron in complex with spliced exons and two additional structures that examine the role of active-site metal ions during the second step of RNA splicing. These structures reveal a relaxed active site, in which direct metal coordination by the exons is lost after ligation, while other tertiary interactions are retained between the exon and the intron. Consistent with these structural observations, kinetic and thermodynamic measurements show that the scissile phosphate makes direct contact with metals in the ground state before exon ligation and in the transition state, but not after exon ligation. Despite no direct exonic interactions and even in the absence of the scissile phosphate, two metal ions remain bound within the active site. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.

  11. Kinetic Study of the Active Site Structure of β-Amylase from Bacillus cereus var. mycoides.

    PubMed

    Nitta, Y; Shirakawa, M; Takasaki, Y

    1996-01-01

    The subsite affinities of the active site of β-amylase from Bacillus cereus var. mycoides were evaluated based on Hiromi's theory, using (14)C-radiolabeled maltooligosaccharides as substrate. It was estimated that the active site consisted of six subsites, and all subsite affinities could be evaluated. The active site had a common subsite arrangement with those of β -amylases from soybean and wheat bran. The intrinsic breakdown rate constant of α-1,4 glucosidic linkage (kint) was five to seven times as large as those of the other enzymes.From the pH dependence of log[k0/Km], pK values of two functional ionizable groups were pK1 =4.0 and pK2 = 8.4. The pK values were 0.5-0.6 units for pK1 and 0.2-0.3 units for pK2 larger than those of the other enzymes. For the affinity-labeling of this enzyme by 2, 3 epoxypropyl α-D-glucopyranoside (α-EPG), the binding affinity of α-EPG was 1-1.6kcal/mol larger than those of the other β-amylases.

  12. Identification of active site residues of Fenugreek β-amylase: chemical modification and in silico approach.

    PubMed

    Srivastava, Garima; Singh, Vinay K; Kayastha, Arvind M

    2014-10-01

    The amino acid sequence of Fenugreek β-amylase is not available in protein data bank. Therefore, an attempt has been made to identify the catalytic amino acid residues of enzyme by employing studies of pH dependence of enzyme catalysis, chemical modification and bioinformatics. Treatment of purified Fenugreek β-amylase with EDAC in presence of glycine methyl ester and sulfhydryl group specific reagents (IAA, NEM and p-CMB), followed a pseudo first-order kinetics and resulted in effective inactivation of enzyme. The reaction with EDAC in presence of NTEE (3-nitro-l-tyrosine ethylester) resulted into modification of two carboxyl groups per molecule of enzyme and presence of one accessible sulfhydryl group at the active site, per molecule of enzyme was ascertained by titration with DTNB. The above results were supported by the prevention of inactivation of enzyme in presence of substrate. Based on MALDI-TOF analysis of purified Fenugreek β-amylase and MASCOT search, β-amylase of Medicago sativa was found to be the best match. To further confirm the amino acid involved in catalysis, homology modelling of β-amylase of M. sativa was performed. The sequence alignment, superimposition of template and target models, along with study of interactions involved in docking of sucrose and maltose at the active site, led to identification of Glu187, Glu381 and Cys344 as active site residues.

  13. Active site structure and catalytic mechanism of phosphodiesterase for degradation of intracellular second messengers

    NASA Astrophysics Data System (ADS)

    Zhan, Chang-Guo

    2002-03-01

    Phosphodiesterases are clinical targets for a variety of biological disorders, because this superfamily of enzymes regulate intracellular concentration of cyclic nucleotides that serve as the second messengers playing a critical role in a variety of physiological processes. Understanding structure and mechanism of a phosphodiesterase will provide a solid basis for rational design of the more efficient therapeutics. Although a three-dimensional X-ray crystal structure of the catalytic domain of human phosphodiesterase 4B2B was recently reported, it was uncertain whether a critical bridging ligand in the active site is a water molecule or a hydroxide ion. The identity of this bridging ligand has been determined by performing first-principles quantum chemical calculations on models of the active site. All the results obtained indicate that this critical bridging ligand in the active site of the reported X-ray crystal structure is a hydroxide ion, rather than a water molecule, expected to serve as the nucleophile to initialize the catalytic degradation of the intracellular second messengers.

  14. Aerosol measurements at a high-elevation site: composition, size, and cloud condensation nuclei activity

    SciTech Connect

    Friedman, Beth; Zelenyuk, Alla; Beranek, Josef; Kulkarni, Gourihar R.; Pekour, Mikhail S.; Hallar, Anna G.; McCubbin, Ian; Thornton, Joel A.; Cziczo, D. J.

    2013-12-09

    We present measurements of CCN concentrations and associated aerosol composition and size properties at a high-elevation research site in March 2011. CCN closure and aerosol hygroscopicity were assessed using simplified assumptions of bulk aerosol properties as well as a new method utilizing single particle composition and size to assess the importance of particle mixing state in CCN activation. Free troposphere analysis found no significant difference between the CCN activity of free tropospheric aerosol and boundary layer aerosol at this location. Closure results indicate that using only size and number information leads to adequate prediction, in the majority of cases within 50%, of CCN concentrations, while incorporating the hygroscopicity parameters of the individual aerosol components measured by single particle mass spectrometry adds to the agreement, in most cases within 20%, between predicted and measured CCN concentrations. For high-elevation continental sites, with largely aged aerosol and low amounts of local area emissions, a lack of chemical knowledge and hygroscopicity may not hinder models in predicting CCN concentrations. At sites influenced by fresh emissions or more heterogeneous particle types, single particle composition information may be more useful in predicting CCN concentrations and understanding the importance of particle mixing state on CCN activation.

  15. Alkynol natural products target ALDH2 in cancer cells by irreversible binding to the active site.

    PubMed

    Heydenreuter, Wolfgang; Kunold, Elena; Sieber, Stephan A

    2015-11-11

    Falcarinol and stipudiol are natural products with potent anti-cancer activity found in several vegetables. Here, we use a chemical proteomic strategy to identify ALDH2 as a molecular target of falcarinol in cancer cells and confirm enzyme inhibition via covalent alkylation of the active site. Furthermore, the synthesis of stipudiol led to the observation that ALDH2 exhibits preference for alkynol-based binders. Inhibition of ALDH2 impairs detoxification of reactive aldehydes and limits oxidative stress response, two crucial pathways for cellular viability.

  16. Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

    SciTech Connect

    Sultatos, L.G. Kaushik, R.

    2008-08-01

    The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the B{sub max} and K{sub d} for thioflavin t binding to the peripheral anionic site. However, these changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in B{sub max} did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in B{sub max}, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in K{sub d} represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing K{sub d}, and dimethylphosphorylation of Ser203 decreasing K{sub d}. These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203.

  17. Flexibility Correlation between Active Site Regions Is Conserved across Four AmpC β-Lactamase Enzymes

    PubMed Central

    Brown, Jenna R.; Livesay, Dennis R.

    2015-01-01

    β-lactamases are bacterial enzymes that confer resistance to β-lactam antibiotics, such as penicillins and cephalosporins. There are four classes of β-lactamase enzymes, each with characteristic sequence and structure properties. Enzymes from class A are the most common and have been well characterized across the family; however, less is known about how physicochemical properties vary across the C and D families. In this report, we compare the dynamical properties of four AmpC (class C) β-lactamases using our distance constraint model (DCM). The DCM reliably predicts thermodynamic and mechanical properties in an integrated way. As a consequence, quantitative stability/flexibility relationships (QSFR) can be determined and compared across the whole family. The DCM calculates a large number of QSFR metrics. Perhaps the most useful is the flexibility index (FI), which quantifies flexibility along the enzyme backbone. As typically observed in other systems, FI is well conserved across the four AmpC enzymes. Cooperativity correlation (CC), which quantifies intramolecular couplings within structure, is rarely conserved across protein families; however, it is in AmpC. In particular, the bulk of each structure is composed of a large rigid cluster, punctuated by three flexibly correlated regions located at the active site. These regions include several catalytic residues and the Ω-loop. This evolutionary conservation combined with active their site location strongly suggests that these coupled dynamical modes are important for proper functioning of the enzyme. PMID:26018804

  18. Stochastic analysis of myoelectric temporal signatures for multifunctional single-site activation of prostheses and orthoses.

    PubMed

    Graupe, D; Salahi, J; Zhang, D S

    1985-01-01

    This paper is concerned with a stochastic time-series analysis of the temporal signatures of myoelectric (ME) signals including the determination of model order and sampling rate. The paper considers the use of time-series parameters for the activation of artificial limbs for high-level amputees, of stimulation electrodes or of powered braces for paralysed persons, in several degrees of freedom, from a single or two surface-electrode pairs at locations where considerable ME cross-talk exists. The multifunctional capability from a single site is based on the differences between the time-series (TS) parameters for different muscle activation patterns at the same ME site, these differences being thus used for limb function discrimination via easily trainable muscle activation patterns at the vicinity of the electrode site. Specifically, the analysis is in terms of identifying the AR parameters of a time-domain autoregressive (AR) signature model both for the complete ME spectrum and for parts thereof, and in terms of the autocorrelation of the signal and of the models residual. Determination of sampling rate and of model orders is discussed in detail. It is shown that, using online real-time analysis, differences in the AR time-series parameters can be observed for different trainable patterns of muscle activation, at the same electrode location, even at the same ME power levels, as long as considerable cross-talk exists at the electrode site. These parameter differences can be accentuated if one considers the AR parameters for lower-frequency spectral windows. A case is made in this paper for employing TS analysis to squeeze out information in a distinct but low-level ripple of the low frequency spectrum of the signal. This information tends to be ignored in frequency domain, but is all that the AR parameters care for in TS analysis, since they are not concerned, with a flat-average low-frequency spectrum, i.e., its white-noise-like part, which is the residual term

  19. Flexibility Matters: Cooperative Active Sites in Covalent Organic Framework and Threaded Ionic Polymer.

    PubMed

    Sun, Qi; Aguila, Briana; Perman, Jason; Nguyen, Nicholas; Ma, Shengqian

    2016-12-07

    The combination of two or more reactive centers working in concert on a substrate to facilitate the reaction is now considered state of the art in catalysis, yet there still remains a tremendous challenge. Few heterogeneous systems of this sort have been exploited, as the active sites spatially separated within the rigid framework are usually difficult to cooperate. It is now shown that this roadblock can be surpassed. The underlying principle of the strategy presented here is the integration of catalytic components with excellent flexibility and porous heterogeneous catalysts, as demonstrated by the placement of linear ionic polymers in close proximity to surface Lewis acid active sites anchored on the walls of a covalent organic framework (COF). Using the cycloaddition of the epoxides and CO2 as a model reaction, dramatic activity improvements have been achieved for the composite catalysts in relation to the individual catalytic component. Furthermore, they also clearly outperform the benchmark catalytic systems formed by the combination of the molecular organocatalysts and heterogeneous Lewis acid catalysts, while affording additional recyclability. The extraordinary flexibility and enriched concentration of the catalytically active moieties on linear polymers facilitate the concerted catalysis, thus leading to superior catalytic performance. This work therefore uncovers an entirely new strategy for designing bifunctional catalysts with double-activation behavior and opens a new avenue in the design of multicapable systems that mimic biocatalysis.

  20. Effect of prolonged exposure to organic solvents on the active site environment of subtilisin Carlsberg.

    PubMed

    Bansal, Vibha; Delgado, Yamixa; Fasoli, Ezio; Ferrer, Amaris; Griebenow, Kai; Secundo, Francesco; Barletta, Gabriel L

    2010-06-01

    The potential of enzyme catalysis as a tool for organic synthesis is nowadays indisputable, as is the fact that organic solvents affect an enzyme's activity, selectivity and stability. Moreover, it was recently realized that an enzyme's initial activity is substantially decreased after prolonged exposure to organic media, an effect that further hampers their potential as catalysts for organic synthesis. Regrettably, the mechanistic reasons for these effects are still debatable. In the present study we have made an attempt to explain the reasons behind the partial loss of enzyme activity on prolonged exposure to organic solvents. Fluorescence spectroscopic studies of the serine protease subtilisin Carlsberg chemically modified with polyethylene glycol (PEG-SC) and inhibited with a Dancyl fluorophore, and dissolved in two organic solvents (acetonitrile and 1,4-dioxane) indicate that when the enzyme is initially introduced into these solvents, the active site environment is similar to that in water; however prolonged exposure to the organic medium causes this environment to resemble that of the solvent in which the enzyme is dissolved. Furthermore, kinetic studies show a reduction on both V(max) and K(M) as a result of prolonged exposure to the solvents. One interpretation of these results is that during this prolonged exposure to organic solvents the active-site fluorescent label inhibitor adopts a different binding conformation. Extrapolating this to an enzymatic reaction we argue that substrates bind in a less catalytically favorable conformation after the enzyme has been exposed to organic media for several hours.

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

    SciTech Connect

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

    1991-04-01

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

  2. Molecular dynamics simulations of Zika virus NS3 helicase: Insights into RNA binding site activity.

    PubMed

    Mottin, Melina; Braga, Rodolpho C; da Silva, Roosevelt A; Silva, Joao H Martins da; Perryman, Alexander L; Ekins, Sean; Andrade, Carolina Horta

    2017-03-21

    America is still suffering wit