Low dielectric response in enzyme active site

PubMed Central

Mertz, Edward L.; Krishtalik, Lev I.

2000-01-01

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

Well-Defined Metal-O6 in Metal-Catecholates as a Novel Active Site for Oxygen Electroreduction.

PubMed

Liu, Xuan-He; Hu, Wei-Li; Jiang, Wen-Jie; Yang, Ya-Wen; Niu, Shuai; Sun, Bing; Wu, Jing; Hu, Jin-Song

2017-08-30

Metal-nitrogen coordination sites, M-N x (M = Fe, Co, Ni, etc.), have shown great potential to replace platinum group materials as electrocatalysts for oxygen reduction reaction (ORR). However, the real active site in M-N x is still vague to date due to their complicated structure and composition. It is therefore highly desirable but challenging to develop ORR catalysts with novel and clear active sites, which could meet the needs of comprehensive understanding of structure-function relationships and explore new cost-effective and efficient ORR electrocatalysts. Herein, well-defined M-O 6 coordination in metal-catecholates (M-CATs, M = Ni or Co) is discovered to be catalytically active for ORR via a four-electron-dominated pathway. In view of no pyrolysis involved and unambiguous crystalline structure of M-CATs, the M-O 6 octahedral coordination site with distinct structure is determined as a new type of active site for ORR. These findings extend the scope of metal-nonmetal coordination as an active site for ORR and pave a way for bottom-up design of novel electrocatalysts containing M-O 6 coordination.

Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buhrman, Greg; O; #8242

2012-09-17

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the 'off' and 'on' allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond themore » active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.« less

Restoration of areas degraded by alluvial sand mining: use of soil microbiological activity and plant biomass growth to assess evolution of restored riparian vegetation.

PubMed

Venson, Graziela R; Marenzi, Rosemeri C; Almeida, Tito César M; Deschamps-Schmidt, Alexandre; Testolin, Renan C; Rörig, Leonardo R; Radetski, Claudemir M

2017-03-01

River or alluvial sand mining is causing a variety of environmental problems in the Itajaí-açú river basin in Santa Catarina State (south of Brazil). When this type of commercial activity degrades areas around rivers, environmental restoration programs need to be executed. In this context, the aim of this study was to assess the evolution of a restored riparian forest based on data on the soil microbial activity and plant biomass growth. A reference site and three sites with soil degradation were studied over a 3-year period. Five campaigns were performed to determine the hydrolysis of the soil enzyme fluorescein diacetate (FDA), and the biomass productivity was determined at the end of the studied period. The variation in the enzyme activity for the different campaigns at each site was low, but this parameter did differ significantly according to the site. Well-managed sites showed the highest biomass productivity, and this, in turn, showed a strong positive correlation with soil enzyme activity. In conclusion, soil enzyme activity could form the basis for monitoring and the early prediction of the success of vegetal restoration programs, since responses at the higher level of biological organization take longer, inhibiting the assessment of the project within an acceptable time frame.

Enzyme Active Site Interactions by Raman/FTIR, NMR, and Ab Initio Calculations

PubMed Central

Deng, Hua

2017-01-01

Characterization of enzyme active site structure and interactions at high resolution is important for the understanding of the enzyme catalysis. Vibrational frequency and NMR chemical shift measurements of enzyme-bound ligands are often used for such purpose when X-ray structures are not available or when higher resolution active site structures are desired. This review is focused on how ab initio calculations may be integrated with vibrational and NMR chemical shift measurements to quantitatively determine high-resolution ligand structures (up to 0.001 Å for bond length and 0.01 Å for hydrogen bonding distance) and how interaction energies between bound ligand and its surroundings at the active site may be determined. Quantitative characterization of substrate ionic states, bond polarizations, tautomeric forms, conformational changes and its interactions with surroundings in enzyme complexes that mimic ground state or transition state can provide snapshots for visualizing the substrate structural evolution along enzyme-catalyzed reaction pathway. Our results have shown that the integration of spectroscopic studies with theoretical computation greatly enhances our ability to interpret experimental data and significantly increases the reliability of the theoretical analysis. PMID:24018325

Specification of unique Pit-1 activity in the hGH locus control region

PubMed Central

Shewchuk, Brian M.; Liebhaber, Stephen A.; Cooke, Nancy E.

2002-01-01

The human GH (hGH) gene cluster is regulated by a remote 5′ locus control region (LCR). HSI, an LCR component located 14.5 kb 5′ to the hGH-N promoter, constitutes the primary determinant of high-level hGH-N activation in pituitary somatotropes. HSI encompasses an array of three binding sites for the pituitary-specific POU homeodomain factor Pit-1. In the present report we demonstrate that all three Pit-1 sites in the HSI array contribute to LCR activity in vivo. Furthermore, these three sites as a unit are fully sufficient for position-independent and somatotrope-restricted hGH-N transgene activation. In contrast, the hGH-N transgene is not activated by Pit-1 sites native to either the hGH-N or rat (r)GH gene promoters. These findings suggest that the structures of the Pit-1 binding sites at HSI specify distinct chromatin-dependent activities essential for LCR-mediated activation of hGH in the developing pituitary. PMID:12189206

An approach to functionally relevant clustering of the protein universe: Active site profile‐based clustering of protein structures and sequences

PubMed Central

Knutson, Stacy T.; Westwood, Brian M.; Leuthaeuser, Janelle B.; Turner, Brandon E.; Nguyendac, Don; Shea, Gabrielle; Kumar, Kiran; Hayden, Julia D.; Harper, Angela F.; Brown, Shoshana D.; Morris, John H.; Ferrin, Thomas E.; Babbitt, Patricia C.

2017-01-01

Abstract Protein function identification remains a significant problem. Solving this problem at the molecular functional level would allow mechanistic determinant identification—amino acids that distinguish details between functional families within a superfamily. Active site profiling was developed to identify mechanistic determinants. DASP and DASP2 were developed as tools to search sequence databases using active site profiling. Here, TuLIP (Two‐Level Iterative clustering Process) is introduced as an iterative, divisive clustering process that utilizes active site profiling to separate structurally characterized superfamily members into functionally relevant clusters. Underlying TuLIP is the observation that functionally relevant families (curated by Structure‐Function Linkage Database, SFLD) self‐identify in DASP2 searches; clusters containing multiple functional families do not. Each TuLIP iteration produces candidate clusters, each evaluated to determine if it self‐identifies using DASP2. If so, it is deemed a functionally relevant group. Divisive clustering continues until each structure is either a functionally relevant group member or a singlet. TuLIP is validated on enolase and glutathione transferase structures, superfamilies well‐curated by SFLD. Correlation is strong; small numbers of structures prevent statistically significant analysis. TuLIP‐identified enolase clusters are used in DASP2 GenBank searches to identify sequences sharing functional site features. Analysis shows a true positive rate of 96%, false negative rate of 4%, and maximum false positive rate of 4%. F‐measure and performance analysis on the enolase search results and comparison to GEMMA and SCI‐PHY demonstrate that TuLIP avoids the over‐division problem of these methods. Mechanistic determinants for enolase families are evaluated and shown to correlate well with literature results. PMID:28054422

An approach to functionally relevant clustering of the protein universe: Active site profile-based clustering of protein structures and sequences.

PubMed

Knutson, Stacy T; Westwood, Brian M; Leuthaeuser, Janelle B; Turner, Brandon E; Nguyendac, Don; Shea, Gabrielle; Kumar, Kiran; Hayden, Julia D; Harper, Angela F; Brown, Shoshana D; Morris, John H; Ferrin, Thomas E; Babbitt, Patricia C; Fetrow, Jacquelyn S

2017-04-01

Protein function identification remains a significant problem. Solving this problem at the molecular functional level would allow mechanistic determinant identification-amino acids that distinguish details between functional families within a superfamily. Active site profiling was developed to identify mechanistic determinants. DASP and DASP2 were developed as tools to search sequence databases using active site profiling. Here, TuLIP (Two-Level Iterative clustering Process) is introduced as an iterative, divisive clustering process that utilizes active site profiling to separate structurally characterized superfamily members into functionally relevant clusters. Underlying TuLIP is the observation that functionally relevant families (curated by Structure-Function Linkage Database, SFLD) self-identify in DASP2 searches; clusters containing multiple functional families do not. Each TuLIP iteration produces candidate clusters, each evaluated to determine if it self-identifies using DASP2. If so, it is deemed a functionally relevant group. Divisive clustering continues until each structure is either a functionally relevant group member or a singlet. TuLIP is validated on enolase and glutathione transferase structures, superfamilies well-curated by SFLD. Correlation is strong; small numbers of structures prevent statistically significant analysis. TuLIP-identified enolase clusters are used in DASP2 GenBank searches to identify sequences sharing functional site features. Analysis shows a true positive rate of 96%, false negative rate of 4%, and maximum false positive rate of 4%. F-measure and performance analysis on the enolase search results and comparison to GEMMA and SCI-PHY demonstrate that TuLIP avoids the over-division problem of these methods. Mechanistic determinants for enolase families are evaluated and shown to correlate well with literature results. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

Dynamic Perturbation of the Active Site Determines Reversible Thermal Inactivation in Glycoside Hydrolase Family 12.

PubMed

Jiang, Xukai; Li, Wen; Chen, Guanjun; Wang, Lushan

2017-02-27

The temperature dependence of enzyme catalysis is highly debated. Specifically, how high temperatures induce enzyme inactivation has broad implications for both fundamental and applied science. Here, we explored the mechanism of the reversible thermal inactivation in glycoside hydrolase family 12 (GH12) using comparative molecular dynamics simulations. First, we investigated the distribution of structural flexibility over the enzyme and found that the active site was the general thermal-sensitive region in GH12 cellulases. The dynamic perturbation of the active site before enzyme denaturation was explored through principal-component analysis, which indicated that variations in the collective motion and conformational ensemble of the active site may precisely correspond to enzyme transition from its active form to the inactive form. Furthermore, the degree of dynamic perturbation of the active site was found to be negatively correlated with the melting temperatures of GH12 enzymes, further proving the importance of the dynamic stability of the active site. Additionally, analysis of the residue-interaction network revealed that the active site in thermophilic enzyme was capable of forming additional contacts with other amino acids than those observed in the mesophilic enzyme. These interactions are likely the key mechanisms underlying the differences in rigidity of the active site. These findings provide further biophysical insights into the reversible thermal inactivation of enzymes and potential applications in future protein engineering.

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Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-10

... disasters. This information is required to determine that the infrastructure of the site supports the...; Request for the Site Inspection and Landowners Authorization/Ingress-Egress Agreement AGENCY: Federal...-1), Request for the Site Inspection; FEMA Form 010-0-10 (formerly 90-31), Landowner's Authorization...

Quantitative assessment of groundwater quality using a biological indicator: some preliminary observations.

PubMed

Pfeil, R M; Venkat, J A; Plimmer, J R; Sham, S; Davis, K; Nair, P P

1994-02-01

The genotoxicity of groundwater was evaluated, using a novel application of the SOS microplate assay (SOSMA). Organic residues were extracted from groundwater samples from Maryland, Pennsylvania, and Delaware by using C-18 bonded silica solid phase extraction tubes. Total organic carbon content (TOC) of water samples was also determined. The genotoxicity of the extracts was determined by the SOSMA. Relative activity (RA) as determined by the SOSMA is a quantitative measure of genotoxicity based on a comparison to the activity of the mutagen, 4-nitroquinoline oxide. Low levels of RA (about 2x background) were detected in waters from sites within these states. There was considerable temporal and spatial variation in the observed RA, but no definite patterns were observed in the variation. Between sampling sites there was a positive correlation between RA and TOC; however, this relationship appeared to be reversed occasionally within a sampling site. The extraction and bioassay methods provide an easy and relatively inexpensive means of determining water quality.

Increasing the structural coverage of tuberculosis drug targets.

PubMed

Baugh, Loren; Phan, Isabelle; Begley, Darren W; Clifton, Matthew C; Armour, Brianna; Dranow, David M; Taylor, Brandy M; Muruthi, Marvin M; Abendroth, Jan; Fairman, James W; Fox, David; Dieterich, Shellie H; Staker, Bart L; Gardberg, Anna S; Choi, Ryan; Hewitt, Stephen N; Napuli, Alberto J; Myers, Janette; Barrett, Lynn K; Zhang, Yang; Ferrell, Micah; Mundt, Elizabeth; Thompkins, Katie; Tran, Ngoc; Lyons-Abbott, Sally; Abramov, Ariel; Sekar, Aarthi; Serbzhinskiy, Dmitri; Lorimer, Don; Buchko, Garry W; Stacy, Robin; Stewart, Lance J; Edwards, Thomas E; Van Voorhis, Wesley C; Myler, Peter J

2015-03-01

High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the Mtb proteome. Here we evaluate an intra-genus "homolog-rescue" strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. Of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structures would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1 Å, >85% side chain identity, and ≥80% PSAPF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

Increasing the structural coverage of tuberculosis drug targets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baugh, Loren; Phan, Isabelle; Begley, Darren W.

High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the Mtb proteome. Here we evaluate an intra-genus “homolog-rescue” strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. We found that of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structuresmore » would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1 Å, >85% side chain identity, and ≥80% PS APF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases.« less

Increasing the structural coverage of tuberculosis drug targets

DOE PAGES

Baugh, Loren; Phan, Isabelle; Begley, Darren W.; ...

2014-12-19

High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the Mtb proteome. Here we evaluate an intra-genus “homolog-rescue” strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. We found that of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structuresmore » would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1 Å, >85% side chain identity, and ≥80% PS APF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases.« less

Increasing the Structural Coverage of Tuberculosis Drug Targets

PubMed Central

Baugh, Loren; Phan, Isabelle; Begley, Darren W.; Clifton, Matthew C.; Armour, Brianna; Dranow, David M.; Taylor, Brandy M.; Muruthi, Marvin M.; Abendroth, Jan; Fairman, James W.; Fox, David; Dieterich, Shellie H.; Staker, Bart L.; Gardberg, Anna S.; Choi, Ryan; Hewitt, Stephen N.; Napuli, Alberto J.; Myers, Janette; Barrett, Lynn K.; Zhang, Yang; Ferrell, Micah; Mundt, Elizabeth; Thompkins, Katie; Tran, Ngoc; Lyons-Abbott, Sally; Abramov, Ariel; Sekar, Aarthi; Serbzhinskiy, Dmitri; Lorimer, Don; Buchko, Garry W.; Stacy, Robin; Stewart, Lance J.; Edwards, Thomas E.; Van Voorhis, Wesley C.; Myler, Peter J.

2015-01-01

High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the Mtb proteome. Here we evaluate an intra-genus “homolog-rescue” strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. Of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structures would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1Å, >85% side chain identity, and ≥80% PSAPF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases. PMID:25613812

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

PubMed Central

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

2016-01-01

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

Hybrid Molecular Structure of the Giant Protease Tripeptidyl Peptidase II

PubMed Central

Chuang, Crystal K.; Rockel, Beate; Seyit, Gönül; Walian, Peter J.; Schönegge, Anne–Marie; Peters, Jürgen; Zwart, Petrus H.; Baumeister, Wolfgang; Jap, Bing K.

2010-01-01

Tripeptidyl peptidase II (TPP II) is the largest known eukaryotic protease (6MDa). It is believed to act downstream of the 26S proteasome cleaving tripeptides from the N– termini of longer peptides and it is implicated in numerous cellular processes. Here we report the structure of Drosophila TPP II determined by a hybrid approach: The structure of the dimer was solved by x–ray crystallography and docked into the three– dimensional map of the holocomplex obtained by single-particle cryo-electron microscopy. The resulting structure reveals the compartmentalization of the active sites inside a system of chambers and suggests the existence of a molecular ruler determining the size of the cleavage products. Furthermore, the structure suggests a model for activation of TPP II involving the relocation of a flexible loop and a repositioning of the active–site serine, coupling it to holocomplex assembly and active site sequestration. PMID:20676100

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 bacteria. Therefore the applicability of on-site enzymatic activity determination as a direct surrogate or proxy parameter for microbiological standard assays and quantification of fecal indicator bacteria (FIB) concentration could not be approved and further research in this field is necessary. Presently we conclude that rapid on-site detection of enzymatic activity is applicable for surface water monitoring and that it constitutes a complementary on-site monitoring parameter with high potential. Selection of the type of measured enzymatic activities has to be done on a catchment-specific basis and further work is needed to learn more about its detailed information characteristics in different habitats. The accomplishment of this method detecting continuous data of enzymatic activity in high temporal resolution caused by a target bacterial member is on the way of becoming a powerful tool for water quality monitoring, health related water quality- and early warning requirements.

Theoretical analysis of the effect of particle size and support on the kinetics of oxygen reduction reaction on platinum nanoparticles

NASA Astrophysics Data System (ADS)

Viswanathan, Venkatasubramanian; Wang, Frank Yi-Fei

2012-07-01

We perform a first-principles based computational analysis of the effect of particle size and support material on the electrocatalytic activity of platinum nanoparticles. Using a mechanism for oxygen reduction that accounts for electric field effects and stabilization from the water layer on the (111) and (100) facets, we show that the model used agrees well with linear sweep voltammetry and rotating ring disk electrode experiments. We find that the per-site activity of the nanoparticle saturates for particles larger than 5 nm and we show that the optimal particle size is in the range of 2.5-3.5 nm, which agrees well with recent experimental work. We examine the effect of support material and show that the perimeter sites on the metal-support interface are important in determining the overall activity of the nanoparticles. We also develop simple geometric estimates for the activity which can be used for determining the activity of other particle shapes and sizes.We perform a first-principles based computational analysis of the effect of particle size and support material on the electrocatalytic activity of platinum nanoparticles. Using a mechanism for oxygen reduction that accounts for electric field effects and stabilization from the water layer on the (111) and (100) facets, we show that the model used agrees well with linear sweep voltammetry and rotating ring disk electrode experiments. We find that the per-site activity of the nanoparticle saturates for particles larger than 5 nm and we show that the optimal particle size is in the range of 2.5-3.5 nm, which agrees well with recent experimental work. We examine the effect of support material and show that the perimeter sites on the metal-support interface are important in determining the overall activity of the nanoparticles. We also develop simple geometric estimates for the activity which can be used for determining the activity of other particle shapes and sizes. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr30572k

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

PubMed Central

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

1987-01-01

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

Spin-orbit effects on the (119)Sn magnetic-shielding tensor in solids: a ZORA/DFT investigation.

PubMed

Alkan, Fahri; Holmes, Sean T; Iuliucci, Robbie J; Mueller, Karl T; Dybowski, Cecil

2016-07-28

Periodic-boundary and cluster calculations of the magnetic-shielding tensors of (119)Sn sites in various co-ordination and stereochemical environments are reported. The results indicate a significant difference between the predicted NMR chemical shifts for tin(ii) sites that exhibit stereochemically-active lone pairs and tin(iv) sites that do not have stereochemically-active lone pairs. The predicted magnetic shieldings determined either with the cluster model treated with the ZORA/Scalar Hamiltonian or with the GIPAW formalism are dependent on the oxidation state and the co-ordination geometry of the tin atom. The inclusion of relativistic effects at the spin-orbit level removes systematic differences in computed magnetic-shielding parameters between tin sites of differing stereochemistries, and brings computed NMR shielding parameters into significant agreement with experimentally-determined chemical-shift principal values. Slight improvement in agreement with experiment is noted in calculations using hybrid exchange-correlation functionals.

The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

PubMed Central

Pettit, Steven C.; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H.

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation. PMID:12477841

The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

PubMed

Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

PubMed Central

Novakovic, Valerie A.; Shi, Jialan; Rasmussen, Jan; Pipe, Steven W.

2015-01-01

Thrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three- to sixfold when soluble fibrin (SF) binds the αIIbβ3 integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two- to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites. PMID:26162408

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eaton, Todd R.; Boston, Andrew M.; Thompson, Anthony B.

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, TiO x domains are titrated on TiO x–SiO 2 selectively with phenylphosphonic acid (PPA). An ex situ method quantifies all fluid-accessible TiO x, whereas an in situ titration during cis-cyclooctene epoxidation provides previously unavailable values for the number of tetrahedral Ti sites on which H 2O 2 activation occurs. We use this method to determine the active sitemore » 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.« less

Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

PubMed Central

Hughes, Samantha J; Tanner, Julian A; Hindley, Alison D; Miller, Andrew D; Gould, Ian R

2003-01-01

Background Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. Results Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. Conclusions The asymmetry uncovered here appears to be a common feature of oligomeric aminoacyl-tRNA synthetases, and may play an important functional role. We suggest a manner in which catalytic efficiency could be improved by LysU operating in an alternating sites mechanism. PMID:12787471

Mechanism of Metal Ion Activation of the Diphtheria Toxin Repressor DtxR

NASA Astrophysics Data System (ADS)

D'Aquino, J. Alejandro; Ringe, Dagmar

2006-08-01

The diphtheria toxin repressor, DtxR, is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear (1 - 3). Calorimetric techniques have demonstrated that while binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 × 10-7, binding site 2 (primary) is a low affinity binding site with a binding constant of 6.3 × 10-4. These two binding sites act independently and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A,C102D), reported here and the previously reported DtxR(H79A) (4) has allowed us to propose a mechanism of metal ion activation for DtxR.

Determination of conformation and orientation of immobilized peptides and proteins at buried interfaces

NASA Astrophysics Data System (ADS)

Shen, Lei; Ulrich, Nathan W.; Mello, Charlene M.; Chen, Zhan

2015-01-01

Surface immobilized peptides/proteins have important applications such as antimicrobial coating and biosensing. We report a study of such peptides/proteins using sum frequency generation vibrational spectroscopy and ATR-FTIR. Immobilization on surfaces via physical adsorption and chemical coupling revealed that structures of chemically immobilized peptides are determined by immobilization sites, chemical environments, and substrate surfaces. In addition, controlling enzyme orientation by engineering the surface immobilization site demonstrated that structures can be well-correlated to measured chemical activity. This research facilitates the development of immobilized peptides/proteins with improved activities by optimizing their surface orientation and structure.

A multi-biomarker approach in cross-transplanted mussels Mytilus galloprovincialis.

PubMed

Serafim, Angela; Lopes, Belisandra; Company, Rui; Cravo, Alexandra; Gomes, Tânia; Sousa, Vânia; Bebianno, Maria João

2011-11-01

The present work integrates the active biomonitoring (ABM) concept in mussels Mytilus galloprovincialis from the South coast of Portugal transplanted during 28 days between two sites with different sources of contamination, and vice versa, in order to assess biological effects in these mussels. For that purpose a multibiomarker approach was used. The suit of biomarkers indicative of metal contamination were metallothioneins (MT) and the enzyme δ-aminolevulinic acid dehydratase (ALAD), for organic contamination mixed function oxidase system (MFO), glutathione-S-transferase (GST) and acetylcholinesterase (AChE), as oxidative stress biomarkers superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and lipid peroxidation (LPO). These biomarkers were used to determine an index to evaluate the stress levels in these two sites. Site A is strongly influenced by metallic contamination, with higher Cu, Cr and Pb in M. galloprovincialis, as well as higher MT levels, antioxidant enzymes activities and LPO concentrations, and lower ALAD activity. In site B organic compounds (PAHs) are prevalent and native mussels show higher activities of the MFO system components and GST. Transplanted mussels had significant alterations in some biomarkers that reflect the type of contaminants present in each site, which demonstrates the primary role of the environment in determining the physiological characteristics of resident mussels. Therefore the application of ABM using a battery of biomarkers turns out to be a useful approach in sites where usually complex mixtures of contaminants occurs. In this study the biomarkers that better differentiate the impact of different contaminants at each site were MT, CYP450, SOD and CAT.

The role of the peripheral benzodiazepine receptor in photodynamic activity of certain pyropheophorbide ether photosensitizers: albumin site II as a surrogate marker for activity.

PubMed

Dougherty, Thomas J; Sumlin, Adam B; Greco, William R; Weishaupt, Kenneth R; Vaughan, Lurine A; Pandey, Ravindra K

2002-07-01

A study has been carried out to define the importance of the peripheral benzodiazepine receptor (PBR) as a binding site for a series of chlorin-type photosensitizers, pyropheophorbide-a ethers, the subject of a previous quantitative structure-activity relationship study by us. The effects of the PBR ligand PK11195 on the photodynamic activity have been determined in vivo for certain members of this series of alkyl-substituted ethers: two of the most active derivatives (hexyl and heptyl), the least active derivative (dodecyl [C12]) and one of intermediate activity (octyl [C8]). The photodynamic therapy (PDT) effect was inhibited by PK11195 for both of the most active derivatives, but no effect on PDT activity was found for the less active C12 or C8 ethers. The inhibitory effects of PK11195 were predicted by the binding of only the active derivatives to the benzodiazepine site on albumin, ie. human serum albumin (HSA)-Site II. Thus, as with certain other types of photosensitizers, it has been demonstrated with this series of pyropheophorbide ethers that in vitro binding to HSA-Site II is a predictor of both optimal in vivo activity and binding to the PBR in vivo.

Active-Site Plasticity Is Essential to Carbapenem Hydrolysis by OXA-58 Class D β-Lactamase of Acinetobacter baumannii.

PubMed

Pratap, Shivendra; Katiki, Madhusudhanarao; Gill, Preet; Kumar, Pravindra; Golemi-Kotra, Dasantila

2016-01-01

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are a subgroup of class D β-lactamases, which are enzymes that hydrolyze β-lactams. They have attracted interest due to the emergence of multidrug-resistant Acinetobacter baumannii, which is not responsive to treatment with carbapenems, the usual antibiotics of choice for this bacterium. Unlike other class D β-lactamases, these enzymes efficiently hydrolyze carbapenem antibiotics. To explore the structural requirements for the catalysis of carbapenems by these enzymes, we determined the crystal structure of the OXA-58 CHDL of A. baumannii following acylation of its active-site serine by a 6α-hydroxymethyl penicillin derivative that is a structural mimetic for a carbapenem. In addition, several point mutation variants of the active site of OXA-58, as identified by the crystal structure analysis, were characterized kinetically. These combined studies confirm the mechanistic relevance of a hydrophobic bridge formed over the active site. This structural feature is suggested to stabilize the hydrolysis-productive acyl-enzyme species formed from the carbapenem substrates of this enzyme. Furthermore, our structural studies provide strong evidence that the hydroxyethyl group of carbapenems samples different orientations in the active sites of CHDLs, and the optimum orientation for catalysis depends on the topology of the active site allowing proper closure of the active site. We propose that CHDLs use the plasticity of the active site to drive the mechanism of carbapenem hydrolysis toward efficiency. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data.

PubMed

Tripathi, Arti; Gupta, Kritika; Khare, Shruti; Jain, Pankaj C; Patel, Siddharth; Kumar, Prasanth; Pulianmackal, Ajai J; Aghera, Nilesh; Varadarajan, Raghavan

2016-11-01

Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95 pdz3 ) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The enviornmental assessment of a contemporary coal mining system

NASA Technical Reports Server (NTRS)

Dutzi, E. J.; Sullivan, P. J.; Hutchinson, C. F.; Stevens, C. M.

1980-01-01

A contemporary underground coal mine in eastern Kentucky was assessed in order to determine potential off-site and on-site environmental impacts associated with the mining system in the given environmental setting. A 4 section, continuous room and pillor mine plan was developed for an appropriate site in eastern Kentucky. Potential environmental impacts were identified, and mitigation costs determined. The major potential environmental impacts were determined to be: acid water drainage from the mine and refuse site, uneven subsidence of the surface as a result of mining activity, and alteration of ground water aquifers in the subsidence zone. In the specific case examined, the costs of environmental impact mitigation to levels prescribed by regulations would not exceed $1/ton of coal mined, and post mining land values would not be affected.

30 CFR 874.17 - AML agency procedures for reclamation projects receiving less than 50 percent government funding.

Code of Federal Regulations, 2010 CFR

2010-07-01

...) Characterize the site in terms of mine drainage, active slides and slide-prone areas, erosion and sedimentation... the site; (iv) Current and historic coal production in the area; and (v) Any known or anticipated interest in mining the site. (2) You must determine the likelihood that nearby or adjacent mining...

30 CFR 874.17 - AML agency procedures for reclamation projects receiving less than 50 percent government funding.

Code of Federal Regulations, 2011 CFR

2011-07-01

...) Characterize the site in terms of mine drainage, active slides and slide-prone areas, erosion and sedimentation... the site; (iv) Current and historic coal production in the area; and (v) Any known or anticipated interest in mining the site. (2) You must determine the likelihood that nearby or adjacent mining...

10 CFR 765.30 - Reimbursement of costs incurred in accordance with a plan for subsequent remedial action.

Code of Federal Regulations, 2013 CFR

2013-01-01

... COSTS OF REMEDIAL ACTION AT ACTIVE URANIUM AND THORIUM PROCESSING SITES Additional Reimbursement... satisfied in full by the licensee, and (2) The total cost of remedial action required at the site, together... at the site, as determined in the approved plan for subsequent remedial action, by the Federal...

30 CFR 874.17 - AML agency procedures for reclamation projects receiving less than 50 percent government funding.

Code of Federal Regulations, 2013 CFR

2013-07-01

...) Characterize the site in terms of mine drainage, active slides and slide-prone areas, erosion and sedimentation... the site; (iv) Current and historic coal production in the area; and (v) Any known or anticipated interest in mining the site. (2) You must determine the likelihood that nearby or adjacent mining...

30 CFR 874.17 - AML agency procedures for reclamation projects receiving less than 50 percent government funding.

Code of Federal Regulations, 2014 CFR

2014-07-01

...) Characterize the site in terms of mine drainage, active slides and slide-prone areas, erosion and sedimentation... the site; (iv) Current and historic coal production in the area; and (v) Any known or anticipated interest in mining the site. (2) You must determine the likelihood that nearby or adjacent mining...

30 CFR 874.17 - AML agency procedures for reclamation projects receiving less than 50 percent government funding.

Code of Federal Regulations, 2012 CFR

2012-07-01

...) Characterize the site in terms of mine drainage, active slides and slide-prone areas, erosion and sedimentation... the site; (iv) Current and historic coal production in the area; and (v) Any known or anticipated interest in mining the site. (2) You must determine the likelihood that nearby or adjacent mining...

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

PubMed

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

2016-07-15

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Predatory fish depletion and recovery potential on Caribbean reefs.

PubMed

Valdivia, Abel; Cox, Courtney Ellen; Bruno, John Francis

2017-03-01

The natural, prehuman abundance of most large predators is unknown because of the lack of historical data and a limited understanding of the natural factors that control their populations. Determining the supportable predator biomass at a given location (that is, the predator carrying capacity) would help managers to optimize protection and would provide site-specific recovery goals. We assess the relationship between predatory reef fish biomass and several anthropogenic and environmental variables at 39 reefs across the Caribbean to (i) estimate their roles determining local predator biomass and (ii) determine site-specific recovery potential if fishing was eliminated. We show that predatory reef fish biomass tends to be higher in marine reserves but is strongly negatively related to human activities, especially coastal development. However, human activities and natural factors, including reef complexity and prey abundance, explain more than 50% of the spatial variation in predator biomass. Comparing site-specific predator carrying capacities to field observations, we infer that current predatory reef fish biomass is 60 to 90% lower than the potential supportable biomass in most sites, even within most marine reserves. We also found that the scope for recovery varies among reefs by at least an order of magnitude. This suggests that we could underestimate unfished biomass at sites that provide ideal conditions for predators or greatly overestimate that of seemingly predator-depleted sites that may have never supported large predator populations because of suboptimal environmental conditions.

Acidity, oxophilicity and hydrogen sticking probability of supported metal catalysts for hydrodeoxygenation process

NASA Astrophysics Data System (ADS)

Lup, A. Ng K.; Abnisa, F.; Daud, W. M. A. W.; Aroua, M. K.

2018-03-01

Hydrodeoxygenation is an oxygen removal process that occurs in the presence of hydrogen and catalysts. This study has shown the importance of acidity, oxophilicity and hydrogen sticking probability of supported metal catalysts in having high hydrodeoxygenation activity and selectivity. These properties are required to ensure the catalyst has high affinity for C-O or C=O bonds and the capability for the adsorption and activation of H2 and O-containing compounds. A theoretical framework of temperature programmed desorption technique was also discussed for the quantitative understanding of these properties. By using NH3-TPD, the nature and abundance of acid sites of catalyst can be determined. By using H2-TPD, the nature and abundance of metallic sites can also be determined. The desorption activation energy could also be determined based on the Redhead analysis of TPD spectra with different heating rates.

BOREAS TGB-12 Rn-222 Activity Data over the NSA

NASA Technical Reports Server (NTRS)

Trumbore, Susan; Hall, Forrest G. (Editor); Conrad, Sara K. (Editor); Sundquist, Eric; Winston, Greg

2000-01-01

The BOREAS TGB-12 team made measurements of soil carbon inventories, carbon concentration in soil gases, and rates of soil respiration at several sites to estimate the rates of carbon accumulation and turnover in each of the major vegetation types. Sampling strategies for soils were designed to take advantage of local fire chronosequences, so that the accumulation of carbon in regrowing mosses could be determined. All the data are used to: (1) calculate the inventory of carbon and nitrogen in moss and mineral soil layers at NSA sites, (2) determine the rates of input and turnover (using both accumulation since the last stand-killing fire and radiocarbon data), and (3) link changes in soil respiration rate to shifts in the C-14 content of soil CO2 to determine the average "age" respired CO2 . These Rn-222 activity data were collected from 15-Nov-1993 to 16-Aug-1994 over the NSA sites. They are useful for determining the rate of gas exchange between soil and the overlying atmosphere. The data in this data set are stored in tabular ASCII files.

Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spannaus, Ralf; Bodem, Jochen, E-mail: Jochen.Bodem@vim.uni-wuerzburg.de

2014-04-15

In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies.more » The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.« less

Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation

NASA Technical Reports Server (NTRS)

D'Alonzo, Richard C.; Selvamurugan, Nagarajan; Karsenty, Gerard; Partridge, Nicola C.

2002-01-01

Previously, we determined that the activator protein-1 (AP-1)-binding site and the runt domain (RD)-binding site and their binding proteins, c-Fos.c-Jun and Cbfa, regulate the collagenase-3 promoter in parathyroid hormone-treated and differentiating osteoblasts. Here we show that Cbfa1 and c-Fos.c-Jun appear to cooperatively bind the RD- and AP-1-binding sites and form ternary structures in vitro. Both in vitro and in vivo co-immunoprecipitation and yeast two-hybrid studies further demonstrate interaction between Cbfa1 with c-Fos and c-Jun in the absence of phosphorylation and without binding to DNA. Additionally, only the runt domain of Cbfa1 was required for interaction with c-Jun and c-Fos. In mammalian cells, overexpression of Cbfa1 enhanced c-Jun activation of AP-1-binding site promoter activity, demonstrating functional interaction. Finally, insertion of base pairs that disrupted the helical phasing between the AP-1- and RD-binding sites also inhibited collagenase-3 promoter activation. Thus, we provide direct evidence that Cbfa1 and c-Fos.c-Jun physically interact and cooperatively bind the AP-1- and RD-binding sites in the collagenase-3 promoter. Moreover, the AP-1- and RD-binding sites appear to be organized in a specific required helical arrangement that facilitates transcription factor interaction and enables promoter activation.

The Crystal Structure of a Quercetin 2,3-Dioxygenase from Bacillus subtilis Suggests Modulation of Enzyme Activity by a Change in the Metal Ion at the Active Site(s)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gopal, B.; Madan, Lalima L.; Betz, Stephen F.

2010-11-10

Common structural motifs, such as the cupin domains, are found in enzymes performing different biochemical functions while retaining a similar active site configuration and structural scaffold. The soil bacterium Bacillus subtilis has 20 cupin genes (0.5% of the total genome) with up to 14% of its genes in the form of doublets, thus making it an attractive system for studying the effects of gene duplication. There are four bicupins in B. subtilis encoded by the genes yvrK, yoaN, yxaG, and ywfC. The gene products of yvrK and yoaN function as oxalate decarboxylases with a manganese ion at the active site(s),more » whereas YwfC is a bacitracin synthetase. Here we present the crystal structure of YxaG, a novel iron-containing quercetin 2,3-dioxygenase with one active site in each cupin domain. Yxag is a dimer, both in solution and in the crystal. The crystal structure shows that the coordination geometry of the Fe ion is different in the two active sites of YxaG. Replacement of the iron at the active site with other metal ions suggests modulation of enzymatic activity in accordance with the Irving-Williams observation on the stability of metal ion complexes. This observation, along with a comparison with the crystal structure of YvrK determined recently, has allowed for a detailed structure-function analysis of the active site, providing clues to the diversification of function in the bicupin family of proteins.« less

Idaho National Laboratory (INL) Site Greenhouse Gas (GHG) Monitoring Plan - 40 CFR 98

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deborah L. Layton; Kimberly Frerichs

2011-12-01

The purpose of this Greenhouse Gas (GHG) Monitoring Plan is to meet the monitoring plan requirements of Title 40 of the Code of Federal Regulations Part 98.3(g)(5). This GHG Monitoring Plan identifies procedures and methodologies used at the Idaho National Laboratory Site (INL Site) to collect data used for GHG emissions calculations and reporting requirements from stationary combustion and other regulated sources in accordance with 40 CFR 98, Subparts A and other applicable subparts. INL Site Contractors determined subpart applicability through the use of a checklist (Appendix A). Each facility/contractor reviews operations to determine which subparts are applicable and themore » results are compiled to determine which subparts are applicable to the INL Site. This plan is applicable to the 40 CFR 98-regulated activities managed by the INL Site contractors: Idaho National Laboratory (INL), Idaho Cleanup Project (ICP), Advanced Mixed Waste Treatment Project (AMWTP), and Naval Reactors Facilities (NRF).« less

Idaho National Laboratory (INL) Site Greenhouse Gas (GHG) Monitoring Plan - 40 CFR 98

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deborah L. Layton; Kimberly Frerichs

2010-07-01

The purpose of this Greenhouse Gas (GHG) Monitoring Plan is to meet the monitoring plan requirements of Title 40 of the Code of Federal Regulations Part 98.3(g)(5). This GHG Monitoring Plan identifies procedures and methodologies used at the Idaho National Laboratory Site (INL Site) to collect data used for GHG emissions calculations and reporting requirements from stationary combustion and other regulated sources in accordance with 40 CFR 98, Subparts A and other applicable subparts. INL Site Contractors determined subpart applicability through the use of a checklist (Appendix A). Each facility/contractor reviews operations to determine which subparts are applicable and themore » results are compiled to determine which subparts are applicable to the INL Site. This plan is applicable to the 40 CFR 98-regulated activities managed by the INL Site contractors: Idaho National Laboratory (INL), Idaho Cleanup Project (ICP), Advanced Mixed Waste Treatment Project (AMWTP), and Naval Reactors Facilities (NRF).« less

Work-site wellness programmes in Sweden: a cross-sectional study of physical activity, self-efficacy, and health.

PubMed

Gånedahl, H; Zsaludek Viklund, P; Carlén, K; Kylberg, E; Ekberg, J

2015-05-01

In Sweden, a work-site wellness programme implies reimbursing some of the expenses for health-promoting activities. Although work-site wellness programmes are readily available in Sweden, a large number of employees elect not to participate. The aim of this study was to investigate the association of physical activity, self-reported general health assessment and self-efficacy with participation in a work-site wellness programme. A cross-sectional study design was used. An online questionnaire was distributed to employees of a manufacturing company with 2500 employees in southwest Sweden. Those who took advantage of the work-site wellness programme assessed their general health as better and had higher assessment of physical activity. The study showed that being enlisted also implies a higher level of physical activity and general health; however, the effect sizes of these correlations were small. Self-efficacy, i.e. perceived behavioural control, was not associated with participation in the work-site wellness programme. However, self-efficacy was correlated with both general health assessment and physical activity. A regression analysis to determine explanatory contributions to the general health assessment score showed no significant contribution from participation in a work-site wellness programme, but was instead explained by perceived behavioural control and physical activity. Given the small effect size of the difference in physical activity between participators and non-participators in the work-site wellness programme, it is probable that only a small proportion of participators changed their health-promoting activities as a result of the work-site wellness programme. Copyright © 2015 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.

DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination.

PubMed

Wei, Leizhen; Nakajima, Satoshi; Böhm, Stefanie; Bernstein, Kara A; Shen, Zhiyuan; Tsang, Michael; Levine, Arthur S; Lan, Li

2015-07-07

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomkinson, B.; Wernstedt, C.; Hellman, U.

1987-11-01

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

Use of magnetic circular dichroism to study dinuclear metallohydrolases and the corresponding biomimetics.

PubMed

Larrabee, James A; Schenk, Gerhard; Mitić, Nataša; Riley, Mark J

2015-09-01

Magnetic circular dichroism (MCD) is a convenient technique for providing structural and mechanistic insight into enzymatic systems in solution. The focus of this review is on aspects of geometric and electronic structure that can be determined by MCD, and how this method can further our understanding of enzymatic mechanisms. Dinuclear Co(II) systems that catalyse hydrolytic reactions were selected to illustrate the approach. These systems all contain active sites with similar structures consisting of two Co(II) ions bridged by one or two carboxylates and a water or hydroxide. In most of these active sites one Co(II) is five-coordinate and one is six-coordinate, with differing binding affinities. It is shown how MCD can be used to determine which binding site--five or six-coordinate--has the greater affinity. Importantly, zero-field-splitting data and magnetic exchange coupling constants may be determined from the temperature and field dependence of MCD data. The relevance of these data to the function of the enzymatic systems is discussed.

Structure sensitivity in the kinetics and the dynamics of CO oxidation over stepped Pd(335) studied by the molecular beam infrared chemiluminescence technique: Determination of working sites during the steady-state reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Uetsuka, H.; Watanabe, K.; Kimpara, H.

Kinetics and dynamics of CO oxidation have been studied on a stepped Pd(335) surface at a steady-state condition and compared with those on flat Pd(111). The infrared (IR) chemiluminescence technique was applied to determine where the active catalytic sites are on the Pd(335) surface. Since the vibrational energy state of the product CO{sub 2} is sensitive to the structures of the reaction sites on Pd surfaces, information about the working reaction sites during the steady-state CO oxidation can be obtained from the IR emission spectra of the product CO{sub 2}. The production rate of CO{sub 2} was higher on Pd(335)more » than on Pd(111), indicating that the steps on the surface enhance the catalytic activity for Co oxidation under the steady-state condition. However, the rate data do not necessarily show the real active sites for the CO + O recombination reaction. At a surface temperature of 850 K, the vibrational Boltzmann temperature (T{sub v}) of the product CO{sub 2} on Pd(335) was quite different from (much lower than) that on Pd(111), although the Pd(335) surface has four-atom wide (111) terraces. The lower T{sub v} value on Pd(335) was similar to that on Pd(110)(1 x 1), indicating that a relatively linear activated CO{sub 2} complex was formed. Therefore, during the steady-state CO oxidation on Pd(335), the reaction does not take place on the (111) terrace sites, but mostly on the step sites at 850 K. On the contrary, as the CO coverage increased at a lower surface temperature and at a high CO/O{sub 2} ratio, the T{sub v} values on Pd(335) tend to approach those on Pd(111), indicating that the contribution of the active sites on the steps is decreased and the working reaction sites shift to the (111) terrace sites.« less

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. IC 50 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Role of apamin-sensitive small conductance calcium-activated potassium currents in long-term cardiac memory in rabbits.

PubMed

Yin, Dechun; Chen, Mu; Yang, Na; Wu, Adonis Z; Xu, Dongzhu; Tsai, Wei-Chung; Yuan, Yuan; Tian, Zhipeng; Chan, Yi-Hsin; Shen, Changyu; Chen, Zhenhui; Lin, Shien-Fong; Weiss, James N; Chen, Peng-Sheng; Everett, Thomas H

2018-05-01

Apamin-sensitive small conductance calcium-activated K current (I KAS ) is up-regulated during ventricular pacing and masks short-term cardiac memory (CM). The purpose of this study was to determine the role of I KAS in long-term CM. CM was created with 3-5 weeks of ventricular pacing and defined by a flat or inverted T wave off pacing. Epicardial optical mapping was performed in both paced and normal ventricles. Action potential duration (APD 80 ) was determined during right atrial pacing. Ventricular stability was tested before and after I KAS blockade. Four paced hearts and 4 normal hearts were used for western blotting and histology. There were no significant differences in either echocardiographic parameters or fibrosis levels between groups. Apamin induced more APD 80 prolongation in CM than in normal ventricles (mean [95% confidence interval]: 9.6% [8.8%-10.5%] vs 3.1% [1.9%-4.3%]; P <.001). Apamin significantly lengthened APD 80 in the CM model at late activation sites, indicating significant I KAS up-regulation at those sites. The CM model also had altered Ca 2+ handling, with the 50% Ca 2+ transient duration and amplitude increased at distal sites compared to a proximal site (near the pacing site). After apamin, the CM model had increased ventricular fibrillation (VF) inducibility (paced vs control: 33/40 (82.5%) vs 7/20 (35%); P <.001) and longer VF durations (124 vs 26 seconds; P <.001). Chronic ventricular pacing increases Ca 2+ transients at late activation sites, which activates I KAS to maintain repolarization reserve. I KAS blockade increases VF vulnerability in chronically paced rabbit ventricles. Copyright © 2018 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

[Toxic effect of DDT, chlordane and water from the Ignacio Ramírez dam (Mexico), on Daphnia magna (Crustacea: Daphnidae)].

PubMed

Martínez-Tabche, L; Romero Solís, M; López López, E; Galar Martínez, M

1999-12-01

Chlorodiphenylnitrichloroethane (DDT) and chlordane (CLO) are currently used in Mexico to control malaria and termites. From 1990 to 1996 a total of 27 ton of DDT and 508 of CLO were imported. We establish a methodology to determine their environmental impact in a Mexican dam (Ignacio Ramírez). The toxic effect of DDT and CLO were evaluated on the o-demethylase (OD) and acethycholinesterase activities (AchA) of the cladoceran Daphnia magna exposed to different concentrations of the insecticides solved in water from three sites. Their effect on the AchA and OD activities, and so the CL50 were used as exposure bioindicators to determine the more polluted sites. The physicochemical characteristics of water and the biodiversity of the dam test sites were considered. The station near the floodgate has toxicity potential because enzymatic activities were modified. We suggest the use of AchA and OD activities measure in the cladoceran to evaluate the toxicity of a water body polluted by organochlorate insecticides.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Unterberger, Claudia; Hanson, Steven; Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester LE1 9HN

Little is known about determinants regulating expression of Mannan-binding lectin associated serine protease-2 (MASP-2), the effector component of the lectin pathway of complement activation. Comparative bioinformatic analysis of the MASP2 promoter regions in human, mouse, and rat, revealed conservation of two putative Stat binding sites, termed StatA and StatB. Site directed mutagenesis specific for these sites was performed. Transcription activity was decreased 5-fold when StatB site was mutated in the wildtype reporter gene construct. Gel retardation and competition assays demonstrated that proteins contained in the nuclear extract prepared from HepG2 specifically bound double-stranded StatB oligonucleotides. Supershift analysis revealed Stat3 tomore » be the major specific binding protein. We conclude that Stat3 binding is important for MASP2 promoter activity.« less

Mechanism of Metal Ion Activation of the Diphtheria Toxin Repressor DtxR

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Aquino,J.; Tetenbaum-Novatt, J.; White, A.

2005-01-01

The diphtheria toxin repressor (DtxR) is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear. Calorimetric techniques have demonstrated that although binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 x 10{sup -7}, binding site 2 (primary) is a low-affinity binding site with amore » binding constant of 6.3 x 10{sup -4}. These two binding sites act in an independent fashion, and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A, C102D), reported here, and the previously reported DtxR(H79A) have allowed us to propose a mechanism of metal activation for DtxR.« less

Yucca Mountain Biological Resources Monitoring Program; Annual report, FY91

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1992-01-01

The US Department of Energy (DOE) is required by the Nuclear Waste Policy Act of 1982 (as amended in 1987) to study and characterize Yucca Mountain as a possible site for a geologic repository for high-level nuclear waste. During site characterization, the DOE will conduct a variety of geotechnical, geochemical, geological, and hydrological studies to determine the suitability of Yucca Mountain as a repository. To ensure that site characterization activities (SCA) do not adversely affect the Yucca Mountain area, an environmental program has been implemented to monitor and mitigate potential impacts and to ensure that activities comply with applicable environmentalmore » regulations. This report describes the activities and accomplishments during fiscal year 1991 (FY91) for six program areas within the Terrestrial Ecosystem component of the YMP environmental program. The six program areas are Site Characterization Activities Effects, Desert Tortoises, Habitat Reclamation, Monitoring and Mitigation, Radiological Monitoring, and Biological Support.« less

Truncated Dual-Cap Nucleation Site Development

NASA Technical Reports Server (NTRS)

Matson, Douglas M.; Sander, Paul J.

2012-01-01

During heterogeneous nucleation within a metastable mushy-zone, several geometries for nucleation site development must be considered. Traditional spherical dual cap and crevice models are compared to a truncated dual cap to determine the activation energy and critical cluster growth kinetics in ternary Fe-Cr-Ni steel alloys. Results of activation energy results indicate that nucleation is more probable at grain boundaries within the solid than at the solid-liquid interface.

The Role of the β5-α11 Loop in the Active-Site Dynamics of Acylated Penicillin-Binding Protein A from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher

Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constantsmore » for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5–α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5–α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.« less

Pseudomonas aeruginosa 4-Amino-4-Deoxychorismate Lyase: Spatial Conservation of an Active Site Tyrosine and Classification of Two Types of Enzyme

PubMed Central

O'Rourke, Patrick E. F.; Eadsforth, Thomas C.; Fyfe, Paul K.; Shepherd, Sharon M.; Hunter, William N.

2011-01-01

4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of Gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å) crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5′-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp 2 to sp 3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of the mechanism. PMID:21935381

Environmental, policy, and cultural factors related to physical activity in African American women.

PubMed

Richter, Donna L; Wilcox, Sara; Greaney, Mary L; Henderson, Karla A; Ainsworth, Barbara E

2002-01-01

Six focus groups were conducted in South Carolina with African American women (n = 42) aged 19-51 years to identify factors that influence physical activity. Transcripts were analyzed using NUD*IST. Cultural influences were seen as more important in determining the type of physical activity than its level. Barriers to and enablers of physical activity were identified in the social and physical environments, as were policy issues affecting physical activity in the community and at the work site. Potential community and work site interventions were suggested. Child care and monetary costs were frequently cited as barriers to physical activity.

Insights into the binding mode of sulphamates and sulphamides to hCA II: crystallographic studies and binding free energy calculations.

PubMed

De Simone, Giuseppina; Langella, Emma; Esposito, Davide; Supuran, Claudiu T; Monti, Simona Maria; Winum, Jean-Yves; Alterio, Vincenzo

2017-12-01

Sulphamate and sulphamide derivatives have been largely investigated as carbonic anhydrase inhibitors (CAIs) by means of different experimental techniques. However, the structural determinants responsible for their different binding mode to the enzyme active site were not clearly defined so far. In this paper, we report the X-ray crystal structure of hCA II in complex with a sulphamate inhibitor incorporating a nitroimidazole moiety. The comparison with the structure of hCA II in complex with its sulphamide analogue revealed that the two inhibitors adopt a completely different binding mode within the hCA II active site. Starting from these results, we performed a theoretical study on sulphamate and sulphamide derivatives, demonstrating that electrostatic interactions with residues within the enzyme active site play a key role in determining their binding conformation. These findings open new perspectives in the design of effective CAIs using the sulphamate and sulphamide zinc binding groups as lead compounds.

Active-site protein dynamics and solvent accessibility in native Achromobacter cycloclastes copper nitrite reductase.

PubMed

Sen, Kakali; Horrell, Sam; Kekilli, Demet; Yong, Chin W; Keal, Thomas W; Atakisi, Hakan; Moreau, David W; Thorne, Robert E; Hough, Michael A; Strange, Richard W

2017-07-01

Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (Asp CAT and His CAT ) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the Asp CAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site 'capping residue' (Ile CAT ), a determinant of ligand binding, are influenced both by temperature and by the protonation state of Asp CAT . A previously unobserved conformation of Ile CAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.

The implications of episodic nonequilibrium fracture-matrix flow on site suitability and total system performance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nitao, J.J.; Buscheck, T.A.; Chesnut, D.A.

1992-04-01

We apply our work on fracture- and matrix-dominated flow to develop a conceptual model of hydrological flow processes in the unsaturated zone at Yucca Mountain. The possibility of fracture-dominated flow is discussed, and various deductions are made on its impact on natural and total system performance, site characterization activities, and site suitability determination.

Logging Options to Minimize Soil Disturbance in the Northern Lake States

Treesearch

Douglas M. Stone

2002-01-01

Forest harvesting is likely to have greater impacts on site productivity than any other activity during the rotation. We determined effects of commercial, winter-logging of four aspen-dominated stands on site disturbance and development of regeneration on clay soils in western Upper Michigan. A large skidder caused deep rutting on 20% of a site in a thinning that...

Chemical Rescue of Enzymes: Proton Transfer in Mutants of Human Carbonic Anhydrase II

PubMed Central

Maupin, C. Mark; Castillo, Norberto; Taraphder, Srabani; Tu, Chingkuang; McKenna, Robert; Silverman, David N.; Voth, Gregory A.

2011-01-01

In human carbonic anhydrase II (HCA II) the mutation of position 64 from histidine to alanine (H64A) disrupts the rate limiting proton transfer (PT) event, resulting in a reduction of the catalytic activity of the enzyme as compared to the wild-type. Potential of mean force (PMF) calculations utilizing the multistate empirical valence bond (MS-EVB) methodology for H64A HCA II give a PT free energy barrier significantly higher than that found in the wild-type enzyme. This high barrier, determined in the absence of exogenous buffer and assuming no additional ionizable residues in the PT pathway, indicates the likelihood of alternate enzyme pathways that utilize either ionizable enzyme residues (self-rescue) and/or exogenous buffers (chemical rescue). It has been shown experimentally that the catalytic activity of H64A HCA II can be chemically rescued to near wild type levels by the addition of the exogenous buffer 4-methylimidazole (4MI). Crystallographic studies have identified two 4MI binding sites, yet site specific mutations intended to disrupt 4MI binding have demonstrated these sites to be non-productive. In the present work MS-EVB simulations show that binding of 4MI near Thr199 in the H64A HCA II mutant, a binding site determined by NMR spectroscopy, results in a viable chemical rescue pathway. Additional viable rescue pathways are also identified where 4MI acts as a proton transport intermediary from the active site to ionizable residues on the rim of the active site, revealing a probable mode of action for the chemical rescue pathway PMID:21452838

An Allosteric Coagonist Model for Propofol Effects on α1β2γ2L γ-Aminobutyric Acid Type A Receptors

PubMed Central

Ruesch, Dirk; Neumann, Elena; Wulf, Hinnerk; Forman, Stuart A.

2011-01-01

Background Propofol produces its major actions via γ-aminobutyric acid type A (GABAA) receptors. At low concentrations, propofol enhances agonist-stimulated GABAA receptor activity, and high propofol concentrations directly activate receptors. Etomidate produces similar effects, and there is convincing evidence that a single class of etomidate sites mediate both agonist modulation and direct GABAA receptor activation. It is unknown if the propofol binding site(s) on GABAA receptors that modulate agonist-induced activity also mediate direct activation. Methods GABAA α1β2γ2L receptors were heterologously expressed in Xenopus oocytes and activity was quantified using voltage clamp electrophysiology. We tested whether propofol and etomidate display the same linkage between agonist modulation and direct activation of GABAA receptors by identifying equi-efficacious drug solutions for direct activation. We then determined whether these drug solutions produce equal modulation of GABA-induced receptor activity. We also measured propofol-dependent direct activation and modulation of low GABA responses. Allosteric coagonist models similar to that established for etomidate, but with variable numbers of propofol sites, were fitted to combined data. Results Solutions of 19 μM propofol and 10 μM etomidate were found to equally activate GABAA receptors. These two drug solutions also produced indistinguishable modulation of GABA-induced receptor activity. Combined electrophysiological data behaved in a manner consistent with allosteric co-agonist models with more than one propofol site. The best fit was observed when the model assumed three equivalent propofol sites. Conclusions Our results support the hypothesis that propofol, like etomidate, acts at GABAA receptor sites mediating both GABA modulation and direct activation. PMID:22104494

Non-active site mutation (Q123A) in New Delhi metallo-β-lactamase (NDM-1) enhanced its enzyme activity.

PubMed

Ali, Abid; Azam, Mohd W; Khan, Asad U

2018-06-01

New Delhi metallo β-lactamase-1 is one of the carbapenemases, causing hydrolysis of almost all β-lactamase antibiotics. Seventeen different NDM variants have been reported so far, they varied in their sequences either by single or multiple amino acid substitutions. Hence, it is important to understand its structural and functional relation. In the earlier studies role of active site residues has been studied but non-active site residues has not studied in detail. Therefore, we have initiated to further comprehend its structure and function relation by mutating some of its non-active site residues. A laboratory mutant of NDM-1 was generated by PCR-based site-directed mutagenesis, replacing Q to A at 123 position. The MICs of imipenem and meropenem for NDM-1 Q123A were found increased by 2 fold as compare to wild type and so the hydrolytic activity was enhanced (Kcat/Km) as compared to NDM-1 wild type. GOLD fitness scores were also found in favour of kinetics data. Secondary structure for α-helical content was determined by Far-UV circular dichroism (CD), which showed significant conformational changes. We conclude a noteworthy role of non-active-site amino acid residues in the catalytic activity of NDM-1. This study also provides an insight of emergence of new variants through natural evolution. Copyright © 2018 Elsevier B.V. All rights reserved.

Monitoring Environmental Recovery at Terminated Produced Water Discharge Sites in Coastal Louisiana Waters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Continental Shelf Associates, Inc.

1999-08-16

This report presents the results of a study of terminated produced water discharge sites in the coastal waters of Louisiana. Environmental recovery at the sites is documented by comparing pre-termination and post-termination (six months and one year) data. Produced water, sediments, and sediment interstitial water samples were analyzed for radionuclides, metals, and hydrocarbons. Benthic infauna were identified from samples collected in the vicinity of the discharge and reference sites. Radium isotope activities were determined in fish and crustacean samples. In addition, an environmental risk assessment is made on the basis of the concentrations of metals and hydrocarbons determined in themore » samples.« less

2013 Annual Site Inspection and Monitoring Report for Uranium Mill Tailings Radiation Control Act Title II Disposal Sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

None

This report, in fulfillment of a license requirement, presents the results of long-term surveillance and maintenance activities conducted by the U.S. Department of Energy (DOE) Office of Legacy Management in 2013 at six uranium mill tailings disposal sites reclaimed under Title II of the Uranium Mill Tailings Radiation Control Act (UMTRCA) of 1978. These activities verified that the UMTRCA Title II disposal sites remain in compliance with license requirements. DOE manages six UMTRCA Title II disposal sites under a general license granted by the U.S. Nuclear Regulatory Commission (NRC) established at Title 10 Code of Federal Regulations Part 40.28. Reclamationmore » and site transition activities continue at other sites, and DOE ultimately expects to manage approximately 27 Title II disposal sites. Long-term surveillance and maintenance activities and services for these disposal sites include inspecting and maintaining the sites; monitoring environmental media and institutional controls; conducting any necessary corrective action; and performing administrative, records, stakeholder services, and other regulatory functions. Annual site inspections and monitoring are conducted in accordance with site-specific long-term surveillance plans (LTSPs) and procedures established by DOE to comply with license requirements. Each site inspection is performed to verify the integrity of visible features at the site; to identify changes or new conditions that may affect the long-term performance of the site; and to determine the need, if any, for maintenance, follow-up inspections, or corrective action. LTSPs and site compliance reports are available online at http://www.lm.doe.gov« less

Adjacent DNA sequences modulate Sox9 transcriptional activation at paired Sox sites in three chondrocyte-specific enhancer elements

PubMed Central

Bridgewater, Laura C.; Walker, Marlan D.; Miller, Gwen C.; Ellison, Trevor A.; Holsinger, L. Daniel; Potter, Jennifer L.; Jackson, Todd L.; Chen, Reuben K.; Winkel, Vicki L.; Zhang, Zhaoping; McKinney, Sandra; de Crombrugghe, Benoit

2003-01-01

Expression of the type XI collagen gene Col11a2 is directed to cartilage by at least three chondrocyte-specific enhancer elements, two in the 5′ region and one in the first intron of the gene. The three enhancers each contain two heptameric sites with homology to the Sox protein-binding consensus sequence. The two sites are separated by 3 or 4 bp and arranged in opposite orientation to each other. Targeted mutational analyses of these three enhancers showed that in the intronic enhancer, as in the other two enhancers, both Sox sites in a pair are essential for enhancer activity. The transcription factor Sox9 binds as a dimer at the paired sites, and the introduction of insertion mutations between the sites demonstrated that physical interactions between the adjacently bound proteins are essential for enhancer activity. Additional mutational analyses demonstrated that although Sox9 binding at the paired Sox sites is necessary for enhancer activity, it alone is not sufficient. Adjacent DNA sequences in each enhancer are also required, and mutation of those sequences can eliminate enhancer activity without preventing Sox9 binding. The data suggest a new model in which adjacently bound proteins affect the DNA bend angle produced by Sox9, which in turn determines whether an active transcriptional enhancer complex is assembled. PMID:12595563

Water-quality characteristics of streams in forested and rural areas of North Carolina

USGS Publications Warehouse

Simmons, Clyde E.; Heath, Ralph C.

1979-01-01

Data collected in North Carolina during 1973-78 from a statewide network of 39 rural sampling sites were used to define unpolluted or baseline stream quality. The basins were 90 to 100 percent forested and, except for the unknown effects of air pollution, were relatively unaffected by man 's activities. Five distinct geochemical zones were delineated across the State. The chemical characteristics of surface waters in each zone are similar. Mean and other statistical values for major dissolved constituents, nutrients, and minor elements in base runoff and storm runoff were determined. Twenty additional rural sites were located in basins where farming activities ranged from 15 to 55 percent of basins ' land area. Data from these 20 sites were used for comparison with data from the 39 unpolluted sites to determine the increase in constituent levels caused by man. For basins where farming activities accounted for 20 or more percent of total land use, phosphorus levels were 2 to 13 times greater than those from the forested basins and several major constituents were 2 to 3 times greater. Concentrations of minor elements were essentially the same in both developed and undeveloped basins. (Kosco-USGS)

The Nudix Hydrolase CDP-Chase, a CDP-Choline Pyrophosphatase, Is an Asymmetric Dimer with Two Distinct Enzymatic Activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duong-Ly, Krisna C.; Gabelli, Sandra B.; Xu, WenLian

2011-09-06

A Nudix enzyme from Bacillus cereus catalyzes the hydrolysis of CDP-choline to produce CMP and phosphocholine. Here, we show that in addition, the enzyme has a 3{prime} {yields} 5{prime} RNA exonuclease activity. The structure of the free enzyme, determined to a 1.8-{angstrom} resolution, shows that the enzyme is an asymmetric dimer. Each monomer consists of two domains, an N-terminal helical domain and a C-terminal Nudix domain. The N-terminal domain is placed relative to the C-terminal domain such as to result in an overall asymmetric arrangement with two distinct catalytic sites: one with an 'enclosed' Nudix pyrophosphatase site and the othermore » with a more open, less-defined cavity. Residues that may be important for determining the asymmetry are conserved among a group of uncharacterized Nudix enzymes from Gram-positive bacteria. Our data support a model where CDP-choline hydrolysis is catalyzed by the enclosed Nudix site and RNA exonuclease activity is catalyzed by the open site. CDP-Chase is the first identified member of a novel Nudix family in which structural asymmetry has a profound effect on the recognition of substrates.« less

Use of Artificial Burrows by Burrowing Owls (Athene cunicularia) at the HAMMER Facility on the U.S. Department of Energy Hanford Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alexander, Amanda K.; Sackschewsky, Michael R.; Duberstein, Corey A.

In 2003 the U.S. Department of Energy (DOE) constructed an Emergency Vehicle Operations Course (EVOC) at the Hazardous Material Management and Emergency Response Training and Education Center (HAMMER) in the southern portion of the Hanford Site. Preliminary surveys during 2001 identified an active burrowing owl (Athene cunicularia) burrow and three burrowing owls within the proposed development area. Burrowing owls were classified as a federal species of concern, a Washington State ?candidate? species, a Washington Department of Fish and Wildlife priority species, and a Hanford Site Biological Resources Management Plan Level III resource. Therefore, the mitigation action plan for the projectmore » included the installation of twenty artificial burrows around EVOC in the spring of 2003. The mitigation plan established a success criterion of five percent annual use of the burrows by owls. In July 2005, a field survey of the EVOC burrow complex was conducted to determine use and demography at each site. Burrow locations were mapped and signs of activity (feces, owl tracks, castings, feathers) were recorded. Out of the twenty burrows, twelve were found to be active. Of the eight inactive burrows three appeared to have been active earlier in the 2005 breeding season. A total of nineteen owls were counted but demography could not be determined. It appears that the EVOC mitigation exceeded burrow use goals during 2005. Continued site monitoring and maintenance, according to mitigation plan guidelines should be conducted as prescribed.« less

Long-term surveillance plan for the Bodo Canyon Disposal Site, Durango, Colorado

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1994-03-01

This long-term surveillance plan (LTSP) for the Durango, Colorado, Uranium Mill Tailings Remedial Action (UMTRA) Project disposal site describes the surveillance activities for the Durango (Bodo Canyon) disposal site, which will be referred to as the disposal site throughout this document. The US Department of Energy (DOE) will carry out these activities to ensure that the disposal site continues to function as designed. This LTSP was prepared as a requirement for acceptance under the US Nuclear Regulatory Commission (NRC) general license for custody and long-term care of residual radioactive materials (RRM). RRMs include tailings and other uranium ore processing wastesmore » still at the site, which the DOE determines to be radioactive. This LTSP is based on the DOE`s Guidance for Implementing the UMTRA Project Long-term Surveillance Program (DOE, 1992).« less

Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K.

PubMed

Rout, Manoj K; Lee, Brian L; Lin, Aiyang; Xiao, Wei; Spyracopoulos, Leo

2018-05-03

The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.

Predatory fish depletion and recovery potential on Caribbean reefs

PubMed Central

Valdivia, Abel; Cox, Courtney Ellen; Bruno, John Francis

2017-01-01

The natural, prehuman abundance of most large predators is unknown because of the lack of historical data and a limited understanding of the natural factors that control their populations. Determining the supportable predator biomass at a given location (that is, the predator carrying capacity) would help managers to optimize protection and would provide site-specific recovery goals. We assess the relationship between predatory reef fish biomass and several anthropogenic and environmental variables at 39 reefs across the Caribbean to (i) estimate their roles determining local predator biomass and (ii) determine site-specific recovery potential if fishing was eliminated. We show that predatory reef fish biomass tends to be higher in marine reserves but is strongly negatively related to human activities, especially coastal development. However, human activities and natural factors, including reef complexity and prey abundance, explain more than 50% of the spatial variation in predator biomass. Comparing site-specific predator carrying capacities to field observations, we infer that current predatory reef fish biomass is 60 to 90% lower than the potential supportable biomass in most sites, even within most marine reserves. We also found that the scope for recovery varies among reefs by at least an order of magnitude. This suggests that we could underestimate unfished biomass at sites that provide ideal conditions for predators or greatly overestimate that of seemingly predator-depleted sites that may have never supported large predator populations because of suboptimal environmental conditions. PMID:28275730

An approach for recreation suitability analysis to recreation planning in Gölcük Nature Park.

PubMed

Gül, Atila; Orücü, M Kamil; Karaca, Oznur

2006-05-01

Gölcük Nature Park (GNP) is an area protected by law in Turkey. It is an important nature park with rich flora, fauna, geomorphologic forms, landscape features, and recreational potential in the region. However, GNP does not have a recreation management plan. The purpose of this study was to determine the actual natural, cultural, and visual resources of GNP, determine the most suitable recreational sites with multiple factors, evaluate the demands and tendencies of visitors, and suggest recreational activities and facilities for the most suitable sites of GNP. However, it was also conceived as leading to a recreational plan and design of GNP in the future and identifying the entire appropriate and current data of GNP with the creation of various maps. This study used multifactor analysis to determine the most suitable recreation sites of GNP. Used recreation factors were established including degree of slope, proximity to water resources, accessibility, elevation, vegetation, soil, climate, aspect, current cultural facilities, visual values, and some limiting factors in accordance with the characteristics of GNP. Weighting and suitability values of factors were determined by 30 local expert surveys. All obtained data were evaluated and integrated in the Geographical Information Systems base. Obtained maps were overlapped. Thus, recreational suitability zones map were created manually. However, the demands and behaviours from visitor surveys in GNP were focused on the most suitable recreation sites of the park. Finally, 10% of GNP was identified as the most suitable sites for recreational use. Various recreational facilities and activities (including picnicking, sports facilities and playgrounds, camping sites, walking paths, food and local outlets, etc.) were recommended for nine of the most suitable areas on the proposed recreational map.

A comparison of on-line and off-line bioaerosol measurements at a biowaste site.

PubMed

Feeney, Patrick; Rodríguez, Santiago Fernández; Molina, Rafael; McGillicuddy, Eoin; Hellebust, Stig; Quirke, Michael; Daly, Shane; O'Connor, David; Sodeau, John

2018-06-01

An air measurement campaign was carried out at a green-waste composting site in the South of Ireland during Spring 2016. The aim was to quantify and identify the levels of Primary Biological Aerosol Particles (PBAP) that were present using the traditional off-line, impaction/optical microscopy method alongside an on-line, spectroscopic approach termed WIBS (Wideband Integrated Bioaerosol Sensor), which can provide number concentrations, sizes and "shapes" of airborne PBAP in real-time by use of Light Induced Fluorescence (LIF). The results from the two techniques were compared in order to validate the use of the spectroscopic method for determining the releases of the wide-range of PBAP present there as a function of site activity and meteorological conditions. The seven-day monitoring period undertaken was much longer than any real-time studies that have been previously performed and allowed due comparison between weekday (working) activities at the site and weekend (closed) releases. The time-span also allowed relationships between site activities like turning, agitation or waste delivery and the WIBS data to be determined in a quantitative manner. This information cannot be obtained with the Andersen Sampling methods generally employed at green-waste management sites. Furthermore, few specific bioaerosol types other than Aspergillus fumigatus, are identified using the traditional protocols employed for site licensing purposes. Here though the co-location of WIBS with the impaction instrument made it possible to identify the real-time release behaviour of a specific plant pathogenic spore, Ustilago maydis, present after green-waste deliveries were made by a local distillery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity

USDA-ARS?s Scientific Manuscript database

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...

APPLICATION OF STABLE CARBON AND HYDROGEN ISOTOPIC TECHNIQUES FOR MONITORING BIODEGRADATION OF MTBE IN THE FIELD

EPA Science Inventory

A significant challenge in environmental studies is to determine the onset and extent of MTBE bioremediation at an affected site, which may involve indirect approaches such as microcosm verification of microbial activities at a given site. Stable isotopic fractionation is cha...

Discovery and structure determination of the orphan enzyme isoxanthopterin deaminase .

PubMed

Hall, Richard S; Agarwal, Rakhi; Hitchcock, Daniel; Sauder, J Michael; Burley, Stephen K; Swaminathan, Subramanyam; Raushel, Frank M

2010-05-25

Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a ( gi|44585104 ) and NYSGXRC-9236b ( gi|44611670 ), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 A resolution (Protein Data Bank entry 2PAJ ). This protein folds as a distorted (beta/alpha)(8) barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s(-1), 8.0 muM, and 1.3 x 10(5) M(-1) s(-1) (k(cat), K(m), and k(cat)/K(m), respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9 ). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site residues were used to identify 24 other genes which are predicted to deaminate isoxanthopterin.

Tanshinones that selectively block the collagenase activity of cathepsin K provide a novel class of ectosteric antiresorptive agents for bone.

PubMed

Panwar, Preety; Law, Simon; Jamroz, Andrew; Azizi, Pouya; Zhang, Dongwei; Ciufolini, Marco; Brömme, Dieter

2018-03-01

Attempts to generate active site-directed cathepsin K (CatK) inhibitors for the treatment of osteoporosis have failed because of side effects. We have previously shown that an ectosteric tanshinone CatK inhibitor isolated from Salvia miltiorrhiza blocked, selectively, the collagenase activity of CatK, without affecting the active site and demonstrated its bone-preserving activity in vivo. Here, we have characterize the antiresorptive potential of other tanshinones, which may provide a scaffold for side effect-free CatK inhibitors. Thirty-one tanshinones were tested for their activity against CatK in enzymic and cell-based assays. The inhibitory potency against triple helical and fibrillar collagen degradation was determined in enzymic assays, by scanning electron microscopy and mechanical strength measurements. Human osteoclast assays were used to determine the effects of the inhibitors on bone resorption, its reversibility and osteoclastogenesis. Binding sites were characterized by molecular docking. Twelve compounds showed highly effective anti-collagenase activity and protected collagen against destruction and mechanical instability without inhibiting the hydrolysis of non-collagenous substrates. Six compounds were highly effective in osteoclast bone resorption assays with IC 50 values of <500 nM. None of these tanshinones had effects on cell viability, reversibility of bone resorption inhibition and osteoclastogenesis. The core pharmacophore of the tanshinones appears to be the three-ring system with either a para- or ortho-quinone entity. Our study identified several potent ectosteric antiresorptive CatK inhibitors from the medicinal plant, S. miltiorrhiza, which may avoid side effects seen with active site-directed inhibitors in clinical trials. © 2017 The British Pharmacological Society.

Bioavailability of Pb and Zn from mine tailings as indicated by erythrocyte aminolevulinic acid dehydratase (ALA-D) activity in suckers (Pisces: catostomidae)

USGS Publications Warehouse

Schmitt, Christopher J.; Dwyer, F. James; Finger, Susan E.

1984-01-01

The activity of the erythrocyte enzyme δ-aminolevulinic acid dehydratase (ALA-D) was measured in 35 catostomids (black redhorse, Moxostoma duquesnei; golden redhorse, M. erythrurum; northern hogsucker, Hypentelium nigricans) collected from three sites on a stream contaminated with Pb-, Cd-, and Zn-rich mine tailings and from an uncontaminated site upstream. Enzyme activity was expressed in terms of hemoglobin (Hb), DNA, and protein concentrations; these variables can be determined in the laboratory on once-frozen blood samples. Concentrations of Pb and Zn in blood and of Pb in edible tissues were significantly higher, and ALA-D activity was significantly lower, at all three contaminated sites than upstream. At the most contaminated site, ALA-D activity was 62–67% lower than upstream. Lead concentrations in the edible tissues and in blood were positively correlated (r = 0.80), whereas ALA-D activity was negatively correlated with Pb in blood (r = −0.70) and in edible tissues (r = −0.59). Five statistically significant relations between Pb and Zn in blood and ALA-D activity were determined. The two models that explained the highest percentage (> 74%) of the total variance also included factors related to Hb concentration. All five significant models included negative coefficients for variables that represented Pb in blood and positive coefficients for Zn in blood. The ALA-D assay with results standardized to Hb concentration represents an expedient alternative to the more traditional hematocrit standardization, and the measurement of ALA-D activity by this method can be used to document exposure of fish to environmental Pb.

Quarterly report for the NGA FFCA project, July 1--September 30, 1994

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1994-12-31

Summaries are presented of activities and meetings related to the National Governor`s Association (NGA) Federal Facilities Compliance Act (FFCA) project. The objective of this project is the management of mixed radioactive and hazardous wastes. Activities include: execution of grants to states to fund a review and development of the site treatment plans; determination of interest in a proposed cost model to be developed by EG and G; determination of the major issues that need to be addressed prior to the release of the Draft Site Treatment Plans; and development of a draft Commercial Mixed Waste Issue Paper which was distributedmore » for review and comment.« less

Relevance of Local Flexibility Near the Active Site for Enzymatic Catalysis: Biochemical Characterization and Engineering of Cellulase Cel5A From Bacillus agaradherans.

PubMed

Saavedra, Juan M; Azócar, Mauricio A; Rodríguez, Vida; Ramírez-Sarmiento, César A; Andrews, Barbara A; Asenjo, Juan A; Parra, Loreto P

2018-03-25

Detailed molecular mechanisms underpinning enzymatic reactions are still a central problem in biochemistry. The need for active site flexibility to sustain catalytic activity constitutes a notion of wide acceptance, although its direct influence remains to be fully understood. With the aim of studying the relationship between structural dynamics and enzyme catalysis, the cellulase Cel5A from Bacillus agaradherans is used as a model for in silico comparative analysis with mesophilic and psychrophilic counterparts. Structural features that determine flexibility are related to kinetic and thermodynamic parameters of catalysis. As a result, three specific positions in the vicinity of the active site of Cel5A are selected for protein engineering via site-directed mutagenesis. Three Cel5A variants are generated, N141L, A137Y and I102A/A137Y, showing a concomitant increase in the catalytic activity at low temperatures and a decrease in activation energy and activation enthalpy, similar to cold-active enzymes. These results are interpreted in structural terms by molecular dynamics simulations, showing that disrupting a hydrogen bond network in the vicinity of the active site increases local flexibility. These results provide a structural framework for explaining the changes in thermodynamic parameters observed between homologous enzymes with varying temperature adaptations. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Understanding the determinants of selectivity in drug metabolism through modeling of dextromethorphan oxidation by cytochrome P450

PubMed Central

Oláh, Julianna; Mulholland, Adrian J.; Harvey, Jeremy N.

2011-01-01

Cytochrome P450 enzymes play key roles in the metabolism of the majority of drugs. Improved models for prediction of likely metabolites will contribute to drug development. In this work, two possible metabolic routes (aromatic carbon oxidation and O-demethylation) of dextromethorphan are compared using molecular dynamics (MD) simulations and density functional theory (DFT). The DFT results on a small active site model suggest that both reactions might occur competitively. Docking and MD studies of dextromethorphan in the active site of P450 2D6 show that the dextromethorphan is located close to heme oxygen in a geometry apparently consistent with competitive metabolism. In contrast, calculations of the reaction path in a large protein model [using a hybrid quantum mechanical–molecular mechanics (QM/MM) method] show a very strong preference for O-demethylation, in accordance with experimental results. The aromatic carbon oxidation reaction is predicted to have a high activation energy, due to the active site preventing formation of a favorable transition-state structure. Hence, the QM/MM calculations demonstrate a crucial role of many active site residues in determining reactivity of dextromethorphan in P450 2D6. Beyond substrate binding orientation and reactivity of Compound I, successful metabolite predictions must take into account the detailed mechanism of oxidation in the protein. These results demonstrate the potential of QM/MM methods to investigate specificity in drug metabolism. PMID:21444768

DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination

PubMed Central

Wei, Leizhen; Nakajima, Satoshi; Böhm, Stefanie; Bernstein, Kara A.; Shen, Zhiyuan; Tsang, Michael; Levine, Arthur S.; Lan, Li

2015-01-01

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome. PMID:26100862

From 'omics to otoliths: responses of an estuarine fish to endocrine disrupting compounds across biological scales.

PubMed

Brander, Susanne M; Connon, Richard E; He, Guochun; Hobbs, James A; Smalling, Kelly L; Teh, Swee J; White, J Wilson; Werner, Inge; Denison, Michael S; Cherr, Gary N

2013-01-01

Endocrine disrupting chemicals (EDCs) cause physiological abnormalities and population decline in fishes. However, few studies have linked environmental EDC exposures with responses at multiple tiers of the biological hierarchy, including population-level effects. To this end, we undertook a four-tiered investigation in the impacted San Francisco Bay estuary with the Mississippi silverside (Menidia audens), a small pelagic fish. This approach demonstrated links between different EDC sources and fish responses at different levels of biological organization. First we determined that water from a study site primarily impacted by ranch run-off had only estrogenic activity in vitro, while water sampled from a site receiving a combination of urban, limited ranch run-off, and treated wastewater effluent had both estrogenic and androgenic activity. Secondly, at the molecular level we found that fish had higher mRNA levels for estrogen-responsive genes at the site where only estrogenic activity was detected but relatively lower expression levels where both estrogenic and androgenic EDCs were detected. Thirdly, at the organism level, males at the site exposed to both estrogens and androgens had significantly lower mean gonadal somatic indices, significantly higher incidence of severe testicular necrosis and altered somatic growth relative to the site where only estrogens were detected. Finally, at the population level, the sex ratio was significantly skewed towards males at the site with measured androgenic and estrogenic activity. Our results suggest that mixtures of androgenic and estrogenic EDCs have antagonistic and potentially additive effects depending on the biological scale being assessed, and that mixtures containing androgens and estrogens may produce unexpected effects. In summary, evaluating EDC response at multiple tiers is necessary to determine the source of disruption (lowest scale, i.e. cell line) and what the ecological impact will be (largest scale, i.e. sex ratio).

Functional analysis of iodotyrosine deiodinase from drosophila melanogaster

PubMed Central

Phatarphekar, Abhishek

2016-01-01

Abstract The flavoprotein iodotyrosine deiodinase (IYD) was first discovered in mammals through its ability to salvage iodide from mono‐ and diiodotyrosine, the by‐products of thyroid hormone synthesis. Genomic information indicates that invertebrates contain homologous enzymes although their iodide requirements are unknown. The catalytic domain of IYD from Drosophila melanogaster has now been cloned, expressed and characterized to determine the scope of its potential catalytic function as a model for organisms that are not associated with thyroid hormone production. Little discrimination between iodo‐, bromo‐, and chlorotyrosine was detected. Their affinity for IYD ranges from 0.46 to 0.62 μM (K d) and their efficiency of dehalogenation ranges from 2.4 – 9 x 103 M−1 s−1 (k cat/K m). These values fall within the variations described for IYDs from other organisms for which a physiological function has been confirmed. The relative contribution of three active site residues that coordinate to the amino acid substrates was subsequently determined by mutagenesis of IYD from Drosophila to refine future annotations of genomic and meta‐genomic data for dehalogenation of halotyrosines. Substitution of the active site glutamate to glutamine was most detrimental to catalysis. Alternative substitution of an active site lysine to glutamine affected substrate affinity to the greatest extent but only moderately affected catalytic turnover. Substitution of phenylalanine for an active site tyrosine was least perturbing for binding and catalysis. PMID:27643701

Influence of rete testis fluid deprivation on the kinetic parameters of goat epididymal 5 alpha-reductase.

PubMed

Kelce, W R; Lubis, A M; Braun, W F; Youngquist, R S; Ganjam, V K

1990-01-01

A surgical technique to cannulate the rete testis of the goat was utilized to examine the effects of rete testis fluid (RTF) deprivation on the enzymatic activity of epididymal 5 alpha-reductase. Kinetic techniques were used to determine whether the regional enzymatic effect of RTF deprivation is to decrease the apparent number of 5 alpha-reductase active sites or the catalytic activity of each active site within the epididymal epithelium. Paired comparisons of (Vmax)app and (Km)app values between control and RTF-deprived epididymides indicated that RTF deprivation affected the value of (Vmax)app with no apparent change in the values of (Km)app in caput, corpus, and cauda epididymal regions. We conclude that RTF deprivation in the goat epididymis for 7 days results in a decreased number of apparent 5 alpha-reductase active sites within the epididymal epithelium.

Structural characterization of a non-heme iron active site in zeolites that hydroxylates methane

DOE PAGES

Snyder, Benjamin E. R.; Bottger, Lars H.; Bols, Max L.; ...

2018-04-02

Iron-containing zeolites exhibit unprecedented reactivity in the low-temperature hydroxylation of methane to form methanol. Reactivity occurs at a mononuclear ferrous active site, α-Fe(II), that is activated by N 2O to form the reactive intermediate α-O. This has been defined as an Fe(IV)=O species. Using nuclear resonance vibrational spectroscopy coupled to X-ray absorption spectroscopy, we probe the bonding interaction between the iron center, its zeolite lattice-derived ligands, and the reactive oxygen. α-O is found to contain an unusually strong Fe(IV)=O bond resulting from a constrained coordination geometry enforced by the zeolite lattice. As a result, density functional theory calculations clarify howmore » the experimentally determined geometric structure of the active site leads to an electronic structure that is highly activated to perform H-atom abstraction.« less

Structural characterization of a non-heme iron active site in zeolites that hydroxylates methane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Snyder, Benjamin E. R.; Bottger, Lars H.; Bols, Max L.

Iron-containing zeolites exhibit unprecedented reactivity in the low-temperature hydroxylation of methane to form methanol. Reactivity occurs at a mononuclear ferrous active site, α-Fe(II), that is activated by N 2O to form the reactive intermediate α-O. This has been defined as an Fe(IV)=O species. Using nuclear resonance vibrational spectroscopy coupled to X-ray absorption spectroscopy, we probe the bonding interaction between the iron center, its zeolite lattice-derived ligands, and the reactive oxygen. α-O is found to contain an unusually strong Fe(IV)=O bond resulting from a constrained coordination geometry enforced by the zeolite lattice. As a result, density functional theory calculations clarify howmore » the experimentally determined geometric structure of the active site leads to an electronic structure that is highly activated to perform H-atom abstraction.« less

Uncovering the determinants of a highly perturbed tyrosine pKa in the active site of ketosteroid isomerase.

PubMed

Schwans, Jason P; Sunden, Fanny; Gonzalez, Ana; Tsai, Yingssu; Herschlag, Daniel

2013-11-05

Within the idiosyncratic enzyme active-site environment, side chain and ligand pKa values can be profoundly perturbed relative to their values in aqueous solution. Whereas structural inspection of systems has often attributed perturbed pKa values to dominant contributions from placement near charged groups or within hydrophobic pockets, Tyr57 of a Pseudomonas putida ketosteroid isomerase (KSI) mutant, suggested to have a pKa perturbed by nearly 4 units to 6.3, is situated within a solvent-exposed active site devoid of cationic side chains, metal ions, or cofactors. Extensive comparisons among 45 variants with mutations in and around the KSI active site, along with protein semisynthesis, (13)C NMR spectroscopy, absorbance spectroscopy, and X-ray crystallography, was used to unravel the basis for this perturbed Tyr pKa. The results suggest that the origin of large energetic perturbations are more complex than suggested by visual inspection. For example, the introduction of positively charged residues near Tyr57 raises its pKa rather than lowers it; this effect, and part of the increase in the Tyr pKa from the introduction of nearby anionic groups, arises from accompanying active-site structural rearrangements. Other mutations with large effects also cause structural perturbations or appear to displace a structured water molecule that is part of a stabilizing hydrogen-bond network. Our results lead to a model in which three hydrogen bonds are donated to the stabilized ionized Tyr, with these hydrogen-bond donors, two Tyr side chains, and a water molecule positioned by other side chains and by a water-mediated hydrogen-bond network. These results support the notion that large energetic effects are often the consequence of multiple stabilizing interactions rather than a single dominant interaction. Most generally, this work provides a case study for how extensive and comprehensive comparisons via site-directed mutagenesis in a tight feedback loop with structural analysis can greatly facilitate our understanding of enzyme active-site energetics. The extensive data set provided may also be a valuable resource for those wishing to extensively test computational approaches for determining enzymatic pKa values and energetic effects.

Uncovering the Determinants of a Highly Perturbed Tyrosine pKa in the Active Site of Ketosteroid Isomerase†

PubMed Central

Schwans, Jason P.; Sunden, Fanny; Gonzalez, Ana; Tsai, Yingssu; Herschlag, Daniel

2013-01-01

Within the idiosyncratic enzyme active site environment, side chain and ligand pKa values can be profoundly perturbed relative to their values in aqueous solution. Whereas structural inspection of systems has often attributed perturbed pKa values to dominant contributions from placement near to charged groups or within hydrophobic pockets, Tyr57 of a P. putida ketosteroid isomerase (KSI) mutant, suggested to have a pKa perturbed by nearly 4 units to 6.3, is situated within a solvent-exposed active site devoid of cationic side chains, metal ions, or cofactors. Extensive comparisons among 45 variants with mutations in and around the KSI active site, along with protein semi-synthesis, 13C NMR spectroscopy, absorbance spectroscopy, and x-ray crystallography, was used to unravel the basis for this perturbed Tyr pKa. The results suggest that the origin of large energetic perturbations are more complex than suggested by visual inspection. For example, the introduction of positively charged residues near Tyr57 raises its pKa rather than lowers it; this effect, and part of the increase in the Tyr pKa from introduction of nearby anionic groups arise from accompanying active site structural rearrangements. Other mutations with large effects also cause structural perturbations or appear to displace a structured water molecule that is part of a stabilizing hydrogen bond network. Our results lead to a model in which three hydrogen bonds are donated to the stabilized ionized Tyr, with these hydrogen bond donors, two Tyr side chains and a water molecule, positioned by other side chains and by a water-mediated hydrogen bond network. These results support the notion that large energetic effects are often the consequence of multiple stabilizing interactions, rather than a single dominant interaction. Most generally, this work provides a case study for how extensive and comprehensive comparisons via site-directed mutagenesis in a tight feedback loop with structural analysis can greatly facilitate our understanding of enzyme active site energetics. The extensive dataset provided may also be a valuable resource for those wishing to extensively test computational approaches for determining enzymatic pKa values and energetic effects. PMID:24151972

Oxygen limitation and tissue metabolic potential of the African fish Barbus neumayeri: roles of native habitat and acclimatization

PubMed Central

2011-01-01

Background Oxygen availability in aquatic habitats is a major environmental factor influencing the ecology, behaviour, and physiology of fishes. This study evaluates the contribution of source population and hypoxic acclimatization of the African fish, Barbus neumayeri, in determining growth and tissue metabolic enzyme activities. Individuals were collected from two sites differing dramatically in concentration of dissolved oxygen (DO), Rwembaita Swamp (annual average DO 1.35 mgO2 L-1) and Inlet Stream West (annual average DO 5.58 mgO2 L-1) in Kibale National Park, Uganda, and reciprocally transplanted using a cage experiment in the field, allowing us to maintain individuals under natural conditions of oxygen, food availability, and flow. Fish were maintained under these conditions for four weeks and sampled for growth rate and the activities of phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS), and cytochrome c oxidase (CCO) in four tissues, liver, heart, brain, and skeletal muscle. Results Acclimatization to the low DO site resulted in lower growth rates, lower activities of the aerobic enzyme CCO in heart, and higher activities of the glycolytic enzyme PFK in heart and skeletal muscle. The activity of LDH in liver tissue was correlated with site of origin, being higher in fish collected from a hypoxic habitat, regardless of acclimatization treatment. Conclusions Our results suggest that the influence of site of origin and hypoxic acclimatization in determining enzyme activity differs among enzymes and tissues, but both factors contribute to higher glycolytic capacity and lower aerobic capacity in B. neumayeri under naturally-occurring conditions of oxygen limitation. PMID:21251277

Oxygen limitation and tissue metabolic potential of the African fish Barbus neumayeri: roles of native habitat and acclimatization.

PubMed

Martínez, Mery L; Raynard, Erin L; Rees, Bernard B; Chapman, Lauren J

2011-01-20

Oxygen availability in aquatic habitats is a major environmental factor influencing the ecology, behaviour, and physiology of fishes. This study evaluates the contribution of source population and hypoxic acclimatization of the African fish, Barbus neumayeri, in determining growth and tissue metabolic enzyme activities. Individuals were collected from two sites differing dramatically in concentration of dissolved oxygen (DO), Rwembaita Swamp (annual average DO 1.35 mgO2 L(-1)) and Inlet Stream West (annual average DO 5.58 mgO2 L(-1)) in Kibale National Park, Uganda, and reciprocally transplanted using a cage experiment in the field, allowing us to maintain individuals under natural conditions of oxygen, food availability, and flow. Fish were maintained under these conditions for four weeks and sampled for growth rate and the activities of phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS), and cytochrome c oxidase (CCO) in four tissues, liver, heart, brain, and skeletal muscle. Acclimatization to the low DO site resulted in lower growth rates, lower activities of the aerobic enzyme CCO in heart, and higher activities of the glycolytic enzyme PFK in heart and skeletal muscle. The activity of LDH in liver tissue was correlated with site of origin, being higher in fish collected from a hypoxic habitat, regardless of acclimatization treatment. Our results suggest that the influence of site of origin and hypoxic acclimatization in determining enzyme activity differs among enzymes and tissues, but both factors contribute to higher glycolytic capacity and lower aerobic capacity in B. neumayeri under naturally-occurring conditions of oxygen limitation.

Effects of viscosity and osmotic stress on the reaction of human butyrylcholinesterase with cresyl saligenin phosphate, a toxicant related to aerotoxic syndrome: kinetic and molecular dynamics studies.

PubMed

Masson, Patrick; Lushchekina, Sofya; Schopfer, Lawrence M; Lockridge, Oksana

2013-09-15

CSP (cresyl saligenin phosphate) is an irreversible inhibitor of human BChE (butyrylcholinesterase) that has been involved in the aerotoxic syndrome. Inhibition under pseudo-first-order conditions is biphasic, reflecting a slow equilibrium between two enzyme states E and E'. The elementary constants for CSP inhibition of wild-type BChE and D70G mutant were determined by studying the dependence of inhibition kinetics on viscosity and osmotic pressure. Glycerol and sucrose were used as viscosogens. Phosphorylation by CSP is sensitive to viscosity and is thus strongly diffusion-controlled (kon≈10⁸ M⁻¹·min⁻¹). Bimolecular rate constants (ki) are about equal to kon values, making CSP one of the fastest inhibitors of BChE. Sucrose caused osmotic stress because it is excluded from the active-site gorge. This depleted the active-site gorge of water. Osmotic activation volumes, determined from the dependence of ki on osmotic pressure, showed that water in the gorge of the D70G mutant is more easily depleted than that in wild-type BChE. This demonstrates the importance of the peripheral site residue Asp⁷⁰ in controlling the active-site gorge hydration. MD simulations provided new evidence for differences in the motion of water within the gorge of wild-type and D70G enzymes. The effect of viscosogens/osmolytes provided information on the slow equilibrium E⇌E', indicating that alteration in hydration of a key catalytic residue shifts the equilibrium towards E'. MD simulations showed that glycerol molecules that substitute for water molecules in the enzyme active-site gorge induce a conformational change in the catalytic triad residue His⁴³⁸, leading to the less reactive form E'.

Remedial Action Plan and site design for stabilization of the inactive uranium mill tailings site at Durango, Colorado: Attachment 3, Groundwater hydrology report. Revised final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1991-12-01

The US Environmental Protection Agency (EPA) has established health and environmental protection regulations to correct and prevent groundwater contamination resulting from processing activities at inactive uranium milling sites. According to the Uranium Mill Tailings Radiation Control Act of 1978, (UMTRCA) the US Department of Energy (DOE) is responsible for assessing the inactive uranium processing sites. The DOE has determined this assessment shall include information on hydrogeologic site characterization. The water resources protection strategy that describes how the proposed action will comply with the EPA groundwater protection standards is presented in Attachment 4. Site characterization activities discussed in this section include:more » Characterization of the hydrogeologic environment; characterization of existing groundwater quality; definition of physical and chemical characteristics of the potential contaminant source; and description of local water resources.« less

Protective effects of buckwheat honey on DNA damage induced by hydroxyl radicals.

PubMed

Zhou, Juan; Li, Peng; Cheng, Ni; Gao, Hui; Wang, Bini; Wei, Yahui; Cao, Wei

2012-08-01

To understand the antioxidant properties of buckwheat honeys, we investigated their antioxidant effects on hydroxyl radical-induced DNA breaks in the non-site-specific and site-specific systems, the physicochemical properties, antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical scavenging activity, chelating, and reducing power assays), total phenolic content and individual phenolic acids were also determined. Total phenolic content of buckwheat honeys ranged from 774 to 1694 mg PA/kg, and p-hydroxybenzoic and p-coumaric acids proved to be the main components in buckwheat honeys. All the buckwheat honey samples possess stronger capability to protect DNA in the non-site-specific systems than in the site-specific systems from being damaged by hydroxyl radicals. In the non-site-specific and site-specific system, buckwheat honeys samples prevented ()OH-induced DNA breaks by 21-78% and 5-31% over control value, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

Zampanolide Binding to Tubulin Indicates Cross-Talk of Taxane Site with Colchicine and Nucleotide Sites.

PubMed

Field, Jessica J; Pera, Benet; Gallego, Juan Estévez; Calvo, Enrique; Rodríguez-Salarichs, Javier; Sáez-Calvo, Gonzalo; Zuwerra, Didier; Jordi, Michel; Andreu, José M; Prota, Andrea E; Ménchon, Grégory; Miller, John H; Altmann, Karl-Heinz; Díaz, J Fernando

2018-03-23

The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to β-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 10 5 M -1 in the case of unmodified tubulin to 1.4 ± 0.3 × 10 5 M -1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of β-tubulin.

Geo-Statistical Approach to Estimating Asteroid Exploration Parameters

NASA Technical Reports Server (NTRS)

Lincoln, William; Smith, Jeffrey H.; Weisbin, Charles

2011-01-01

NASA's vision for space exploration calls for a human visit to a near earth asteroid (NEA). Potential human operations at an asteroid include exploring a number of sites and analyzing and collecting multiple surface samples at each site. In this paper two approaches to formulation and scheduling of human exploration activities are compared given uncertain information regarding the asteroid prior to visit. In the first approach a probability model was applied to determine best estimates of mission duration and exploration activities consistent with exploration goals and existing prior data about the expected aggregate terrain information. These estimates were compared to a second approach or baseline plan where activities were constrained to fit within an assumed mission duration. The results compare the number of sites visited, number of samples analyzed per site, and the probability of achieving mission goals related to surface characterization for both cases.

Insights into the Phosphoryl Transfer Catalyzed by cAMP-Dependent Protein Kinase: An X-ray Crystallographic Study of Complexes with Various Metals and Peptide Substrate SP20

PubMed Central

2013-01-01

X-ray structures of several ternary substrate and product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with different bound metal ions. In the PKAc complexes, Mg2+, Ca2+, Sr2+, and Ba2+ metal ions could bind to the active site and facilitate the phosphoryl transfer reaction. ATP and a substrate peptide (SP20) were modified, and the reaction products ADP and the phosphorylated peptide were found trapped in the enzyme active site. Finally, we determined the structure of a pseudo-Michaelis complex containing Mg2+, nonhydrolyzable AMP-PCP (β,γ-methyleneadenosine 5′-triphosphate) and SP20. The product structures together with the pseudo-Michaelis complex provide snapshots of different stages of the phosphorylation reaction. Comparison of these structures reveals conformational, coordination, and hydrogen bonding changes that might occur during the reaction and shed new light on its mechanism, roles of metals, and active site residues. PMID:23672593

Radon soil gas measurements in a geological versatile region as basis to improve the prediction of areas with a high radon potential.

PubMed

Kabrt, Franz; Seidel, Claudia; Baumgartner, Andreas; Friedmann, Harry; Rechberger, Fabian; Schuff, Michael; Maringer, Franz Josef

2014-07-01

With the aim to predict the radon potential by geological data, radon soil gas measurements were made in a selected region in Styria, Austria. This region is characterised by mean indoor radon potentials of 130-280 Bq m(-3) and a high geological diversity. The distribution of the individual measuring sites was selected on the basis of geological aspects and the distribution of area settlements. In this work, the radon soil gas activity concentration and the soil permeability were measured at 100 sites, each with three single measurements. Furthermore, the local dose rate was determined and soil samples were taken at each site to determine the activity concentration of natural radionuclides. During two investigation periods, long-term soil gas radon measurements were made to study the time dependency of the radon activity concentration. All the results will be compared and investigated for correlation among each other to improve the prediction of areas with high radon potential. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Seismic environment of the Burro Flats site, Ventura County, California: a brief, limited literature review

USGS Publications Warehouse

Wentworth, Carl M.; Bonilla, Manuel G.; Buchanan, Jane M.

1969-01-01

A limited review of available literature suggests that the maximum horizontal ground acceleration at the Burro Flats site from earthquakes in the region could range from less than 0.1 to 0.49 g. A magnitude 8 earthquake on the nearby San Andreas fault could produce ground acceleration in the range 0.18 to 0.31 g, and an expectable larger earthquake on that fault could produce larger accelerations. Ground motion from possible smaller but closer earthquakes ranges up to 0.49 g for an earthquake of magnitude 6.5 on the adjacent "Burro Flats fault". Estimation of these accelerations is dependent on determining the geologic environment of the site, the appropriate earthquake magnitudes to be assigned significant faults in that environment, and the attenuation of shaking between the earthquake epicenters and the site. The site lies within a tectonically active region--the historically active San Andreas fault is only 34 miles to the northeast, and lesser faults showing evidence of late Quaternary displacement are located closer to the site. Evidence for youthfulness of these lesser faults varies, and except for the active Newport-Inglewood zone and the Santa Ynez fault, they qualify as possible but as yet-unproven active faults. All known faults with appropriate length to site-distance ratios that are reasonably classed as late Quaternary faults are discussed, and are included as potential earthquake generators. Earthquakes of appropriate magnitude to be assigned to each fault are determined by assuming rupture in one event of half the map length of the fault, and applying relations (determined by several authors) between earthquake magnitude and rupture length in historic events to determine magnitudes. These magnitudes are, for the purposes of this brief review, probably reasonable estimates of the capabilities of each fault, although earthquakes of larger magnitude are possible. Accelerations are then determined by assuming earthquakes of the above determined magnitude placed at the closest point to the site on the fault trace, and applying attenuation curves of three different authors. Considerable uncertainty is inherent in the rough estimates of seismic accelerations made herein, for they are dependent on a chain of judgments, each of which, in itself, is uncertain. Present knowledge of the geology of the region is incomplete, so that geometry and structural relations of the faults are in part uncertain, and much evidence bearing on the youth of the faults has yet to be gathered and evaluated. Estimation of earthquake magnitude is also uncertain, and even assuming that approximate magnitude is known rather than estimated from fault length, estimates of maximum ground acceleration may differ greatly depending on the authority used. Further consideration of ground acceleration at the site might refine the estimates made herein and resolve the apparent contradictions between the authorities cited. Attention to frequency and duration of strong shaking would also be appropriate. This study was undertaken at the request of A. J. Pressesky, Assistant Director for Nuclear Safety, Division of Reactor-Development and Technology, U.S. Atomic Energy Commission, in March, 1969. It is based on a brief review of pertinent literature to which the authors had immediate access during the few weeks (April-May, 1969) available for report preparation. Because the report is limited both in scope and thoroughness, it must be considered no more than a first estimate of the tectonic and seismic environment of the Burro Flats site, and should not be considered sufficient, in itself, as a basis for design. The report is intended, however, to indicate the breadth of inquiry that is necessary in the consideration of ground acceleration at sites in California, and to indicate the incomplete status of geologic mapping and other geologic studies in the region. The report describes the tectonic environment of the Burro Flats site, discusses 10 pertinent faults individually, and presents possible earthquake magnitudes for those faults and resultant potential ground accelerations at the site.

Develop high activity, low cost non-PGM fuel cell electrocatalyst and stable supports

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colon-Mercado, H. R.; Elvington, M. C.; Garcia-Diaz, B. L.

2016-09-28

A unique approach has been developed to probe the non-PGM catalyst active site for the Oxygen Reduction Reaction (ORR) for PEMFC. Iron based functionalities have been engineered into Metallic Organic Framework (MOF) catalysts to evaluate their impact on activity for the ORR. A series of FePhen@MOF catalysts have been synthesized with varying [Fe] to investigate the effect on electrochemical and electrocatalytic properties. The magnitude of the Fe II/III redox couple and the electrochemical surface area are analyzed to determine if there is a correlation between [Fe] and the ORR onset potential and/or the relative number of active sites.

Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability.

PubMed

Kracher, Daniel; Andlar, Martina; Furtmüller, Paul G; Ludwig, Roland

2018-02-02

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa , which is active toward cellulose and soluble β-glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Isolation and characterization of target sequences of the chicken CdxA homeobox gene.

PubMed Central

Margalit, Y; Yarus, S; Shapira, E; Gruenbaum, Y; Fainsod, A

1993-01-01

The DNA binding specificity of the chicken homeodomain protein CDXA was studied. Using a CDXA-glutathione-S-transferase fusion protein, DNA fragments containing the binding site for this protein were isolated. The sources of DNA were oligonucleotides with random sequence and chicken genomic DNA. The DNA fragments isolated were sequenced and tested in DNA binding assays. Sequencing revealed that most DNA fragments are AT rich which is a common feature of homeodomain binding sites. By electrophoretic mobility shift assays it was shown that the different target sequences isolated bind to the CDXA protein with different affinities. The specific sequences bound by the CDXA protein in the genomic fragments isolated, were determined by DNase I footprinting. From the footprinted sequences, the CDXA consensus binding site was determined. The CDXA protein binds the consensus sequence A, A/T, T, A/T, A, T, A/G. The CAUDAL binding site in the ftz promoter is also included in this consensus sequence. When tested, some of the genomic target sequences were capable of enhancing the transcriptional activity of reporter plasmids when introduced into CDXA expressing cells. This study determined the DNA sequence specificity of the CDXA protein and it also shows that this protein can further activate transcription in cells in culture. Images PMID:7909943

French multicenter study involving eight test sites for radiometric determination of activities of 10 antimicrobial agents against Mycobacterium avium complex.

PubMed Central

Rastogi, N; Bauriaud, R M; Bourgoin, A; Carbonnelle, B; Chippaux, C; Gevaudan, M J; Goh, K S; Moinard, D; Roos, P

1995-01-01

The radiometric BACTEC 460-TB methodology has filled an increased need in the screening of a wide range of antimicrobial agents against Mycobacterium avium (MAC) isolates on a patient-to-patient basis. In this context, a multicenter study involving eight test sites across France was performed to determine the MICs of 10 antimicrobial agents for MAC organisms. The aim of the investigation was to compare the in vitro activities of D-cycloserine, ethambutol, ethionamide, rifampin, amikacin, streptomycin, ciprofloxacin, sparfloxacin, clofazimine, and clarithromycin against MAC isolates. All of the test sites received the same clinical isolates of MAC, and the MICs were determined by a common protocol. The overall interlaboratory reproducibility of the MICs within +/- 1 dilution of the modal MICs varied from 79.70 to 100% (mean, 95.2% +/- 2.1%), whereas overall agreement of the MICs among the test sites varied from a mean of 91% +/- 4.1% to a mean of 98 +/- 1.3%. We confirmed that the proposed methodology is easy, accurate, and sufficiently reproducible to be used routinely in a clinical laboratory. Despite variations in the MICs of the same drug among strains, no link between the origin of MAC isolates (from human immunodeficiency virus-positive or -negative patients) and their drug susceptibilities was established. On the basis of the MICs that inhibited 50 and 90% of isolates tested for the drugs used, clarithromycin, clofazimine, ethambutol, and streptomycin were the most uniformly active against MAC; this was followed by amikacin, rifampin, and sparfloxacin. On the other hand, ciprofloxacin, D-cycloserine, and ethionamide showed only marginal in vitro activities. PMID:7793865

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

2017-12-01

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.

Ground water hydrology report: Revision 1, Attachment 3. Final

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1996-12-01

This report presents ground water hydrogeologic activities for the Maybell, Colorado, Uranium Mill Tailings Remedial Action Project site. The Department of Energy has characterized the hydrogeology, water quality, and water resources at the site and determined that the proposed remedial action would comply with the requirements of the EPA ground water protection standards.

29 CFR 825.111 - Determining whether 50 employees are employed within 75 miles.

Code of Federal Regulations, 2013 CFR

2013-07-01

... office manager, etc., from New Jersey to the job site in Ohio, those workers sent from New Jersey... single building, such as an office building, if separate employers conduct activities within the building. For example, an office building with 50 different businesses as tenants will contain 50 sites of...

29 CFR 825.111 - Determining whether 50 employees are employed within 75 miles.

Code of Federal Regulations, 2012 CFR

2012-07-01

... office manager, etc., from New Jersey to the job site in Ohio, those workers sent from New Jersey... single building, such as an office building, if separate employers conduct activities within the building. For example, an office building with 50 different businesses as tenants will contain 50 sites of...

29 CFR 825.111 - Determining whether 50 employees are employed within 75 miles.

Code of Federal Regulations, 2014 CFR

2014-07-01

... office manager, etc., from New Jersey to the job site in Ohio, those workers sent from New Jersey... single building, such as an office building, if separate employers conduct activities within the building. For example, an office building with 50 different businesses as tenants will contain 50 sites of...

29 CFR 825.111 - Determining whether 50 employees are employed within 75 miles.

Code of Federal Regulations, 2011 CFR

2011-07-01

... office manager, etc., from New Jersey to the job site in Ohio, those workers sent from New Jersey... single building, such as an office building, if separate employers conduct activities within the building. For example, an office building with 50 different businesses as tenants will contain 50 sites of...

A Single Base Difference between Pit-1 Binding Sites at the hGH Promoter and Locus Control Region Specifies Distinct Pit-1 Conformations and Functions

PubMed Central

Shewchuk, Brian M.; Ho, Yugong; Liebhaber, Stephen A.; Cooke, Nancy E.

2006-01-01

Activation of the human growth hormone (hGH-N) gene in pituitary somatotropes is mediated by a locus control region (LCR). This LCR is composed of DNase I-hypersensitive sites (HS) located −14.5 kb to −32 kb relative to the hGH-N promoter. HSI, at −14.5 kb, is the dominant determinant of hGH-N expression and is essential for establishment of a 32-kb domain of histone acetylation that encompasses the active hGH locus. This activity is conferred by three binding sites for the POU domain transcription factor Pit-1. These Pit-1 elements are sufficient to activate hGH-N expression in the mouse pituitary. In contrast, Pit-1 sites at the hGH-N promoter are consistently unable to mediate similar activity. In the present study, we demonstrate that the functional difference between the promoter-proximal and the HSI Pit-1 binding sites can be attributed in part to a single base difference. This base affects the conformation of the Pit-1/DNA complex, and reciprocal exchange of the divergent bases between the two sets of Pit-1 elements results in a partial reversal of their transgenic activities. These data support a model in which the Pit-1 binding sites in the hGH LCR allosterically program the bound Pit-1 complex for chromatin activating functions. PMID:16914737

The National Children's Study: Recruitment Outcomes Using an Enhanced Household-Based Approach.

PubMed

Blaisdell, Laura L; Zellner, Jennifer A; King, Alison A; Faustman, Elaine; Wilhelm, Mari; Hudak, Mark L; Annett, Robert D

2016-06-01

Ten National Children's Study (NCS) study locations with diverse demographic characteristics used an enhanced household-based recruitment (EHBR) approach to enroll preconceptional and pregnant women. Study centers used different types and dosages of community outreach and engagement (COE) activities and supplemental strategies. The goal of the study was to determine whether variability in enumeration and recruitment outcomes correlated with study location characteristics or types and dosages of COE activities (number of COE events, number of advance household mailings, total media expenditures, and total COE expenditures). Each of the sites provided data on COE activities, protocol implementation, supplemental recruitment activities, location demographic characteristics, and enumeration/recruitment outcomes. COE activities varied across sites in breadth and scope. Numerous strategies were used, including media advertising, social media, participation in community-wide events, presentations to stakeholders, and creation of advisory boards. Some sites included supplemental recruitment efforts. EHBR sites enrolled 1404 women at the initial pregnancy screening. No significant relationships were found between study location demographic characteristics or between the types and dosages of COE activities and recruitment outcomes. Probability sampling for a long-term study requires a positive image with stakeholders and within communities; this requirement may be especially true for door-to-door recruitment. EHBR sites successfully recruited a representative sample of preconceptional and pregnant women. Sites reported implementing similar COE activities but with varying dosage and cost; however, analyses did not support a benefit of COE strategies on study recruitment. Copyright © 2016 by the American Academy of Pediatrics.

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

PubMed

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

2011-07-01

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

Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase.

PubMed

Appleby, Todd C; Perry, Jason K; Murakami, Eisuke; Barauskas, Ona; Feng, Joy; Cho, Aesop; Fox, David; Wetmore, Diana R; McGrath, Mary E; Ray, Adrian S; Sofia, Michael J; Swaminathan, S; Edwards, Thomas E

2015-02-13

Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site. Copyright © 2015, American Association for the Advancement of Science.

Unraveling the Nature of Active Sites in Ethanol Electro-oxidation by Site-Specific Marking of a Pt Catalyst with Isotope-Labeled 13CO.

PubMed

Farias, Manuel J S; Cheuquepán, William; Tanaka, Auro A; Feliu, Juan M

2018-03-15

This works deals with the identification of preferential site-specific activation at a model Pt surface during a multiproduct reaction. The (110)-type steps of a Pt(332) surface were selectively marked by attaching isotope-labeled 13 CO molecules to them, and ethanol oxidation was probed by in situ Foureir transfrom infrared spectroscopy in order to precisely determine the specific sites at which CO 2 , acetic acid, and acetaldehyde were preferentially formed. The (110) steps were active for splitting the C-C bond, but unexpectedly, we provide evidence that the pathway of CO 2 formation was preferentially activated at (111) terraces, rather than at (110) steps. Acetaldehyde was formed at (111) terraces at potentials comparable to those for CO 2 formation also at (111) terraces, while the acetic acid formation pathway became active only when the (110) steps were released by the oxidation of adsorbed 13 CO, at potentials higher than for the formation of CO 2 at (111) terraces of the stepped surface.

Biochemical indicators of contaminant exposure in birds and turtles of the Great Lakes-St. Lawrence River

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bishop, C.; Trudeau, S.; Kennedy, S.

1995-12-31

Pre-fledgling chicks of tree swallows, double-crested cormorants, herring gulls, common terns and hatchling snapping turtles were collected from contaminated Areas of Concern and reference sites in the Great Lakes and St. Lawrence River to determine the geographic and species variation in biomarker responses. EROD activity in colonial waterbirds was generally an order of magnitude above EROD activity in tree swallows and snapping turtles. Notably, EROD activity in colonial waterbirds did not correlate with organochlorine contamination in livers at one industrialized site suggesting that exposure to other contaminants, possibly PAHs, may be an important factor. Retinol concentrations in cormorants were non-detectablemore » and retinyl palmitate concentrations were equal or greater than those in herring gulls. In tree swallows, there was a significant negative correlation between vitamin A concentration in liver and kidney and EROD activity. In snapping turtles, there was a significant induction in EROD activity and significantly higher cytochrome P450 IAI level in livers from the Great Lakes site relative to a clean inland location. There were no significant differences in porphyrin concentrations between sites.« less

Cations in Octahedral Sites: A Descriptor for Oxygen Electrocatalysis on Transition-Metal Spinels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Chao; Feng, Zhenxing; Scherer, Günther G.

2017-04-10

Exploring efficient and low-cost electrocatalysts for the oxygen-reduction reaction (ORR) and oxygen-evolution reaction (OER) is critical for developing renewable energy technologies such as fuel cells, metal–air batteries, and water electrolyzers. A rational design of a catalyst can be guided by identifying descriptors that determine its activity. Here, a descriptor study on the ORR/OER of spinel oxides is presented. With a series of MnCo2O4, the Mn in octahedral sites is identified as an active site. This finding is then applied to successfully explain the ORR/OER activities of other transition-metal spinels, including MnxCo3-xO4 (x = 2, 2.5, 3), LixMn2O4 (x = 0.7,more » 1), XCo2O4 (X = Co, Ni, Zn), and XFe2O4 (X = Mn, Co, Ni). A general principle is concluded that the eg occupancy of the active cation in the octahedral site is the activity descriptor for the ORR/OER of spinels, consolidating the role of electron orbital filling in metal oxide catalysis.« less

The amino acid sequence around the active-site cysteine and histidine residues of stem bromelain

PubMed Central

Husain, S. S.; Lowe, G.

1970-01-01

Stem bromelain that had been irreversibly inhibited with 1,3-dibromo[2-14C]-acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin three radioactive peptides were isolated chromatographically. The amino acid sequences around the cross-linked cysteine and histidine residues were determined and showed a high degree of homology with those around the active-site cysteine and histidine residues of papain and ficin. PMID:5420046

Determination of TBT in water and sediment samples along the Argentine Atlantic coast.

PubMed

de Waisbaum, R G; Rodriguez, C; Nudelman, N Sbarbati

2010-11-01

Cases of imposex have been reported for some organisms living in areas of the Argentine Atlantic coast. Since this is one of the known effects of the anti-fouling agent tributyltin (TBT), quantitative determinations of organotins in samples of water and sediments collected from sites along the Argentine coast were carried out. Severe cases of imposex were first reported for two gastropod species living in the Mar del Plata area, and determinations of TBT in samples collected from this site gave extremely high values and showed a close correlation between the degree of imposex and TBT concentration. Recent investigations in the area have shown a significant decrease. Surveys were also conducted in sites that exhibit highly irregular coastal profiles to examine the relevance of physical environments. Alarming concentrations of TBT were determined in most of the sites where heavy boat traffic and/or marine activities occur, demonstrating the urgent need for regulations to avoid further input of TBT. Reports from other sites in South America reveal that this should be a subject of regional concern in order to avoid severe damage to the biodiversity of regional marine organisms.

Longitudinal analysis of metalloproteinases, tissue inhibitors of metalloproteinases and clinical parameters in gingival crevicular fluid from periodontitis-affected patients.

PubMed

Pozo, P; Valenzuela, M A; Melej, C; Zaldívar, M; Puente, J; Martínez, B; Gamonal, J

2005-06-01

The aim of this work was to improve the assessment of the periodontal disease status through measurements of extracellular matrix metalloproteinases (MMPs) and their tissular inhibitors (TIMPs) in the gingival crevicular fluid from patients diagnosed with chronic periodontitis. Gingival crevicular fluid samples from patients (n = 13) were taken from 60 sites initially, and from 51 and 41 sites, respectively, 3 and 6 months after scaling and root planing. Gingival crevicular fluid samples were also taken from healthy subjects (n = 11, 24 sites). The presence of MMP-9 and MMP-8 was assessed by zymography and immunowestern blotting, respectively. The actual MMP activity (gelatinase and collagenase) was measured using the fluorogenic substrate assay. TIMP-1 and -2 levels were measured by immunodot blot. The fluorogenic substrate assay determinations showed higher MMP activity in sites with probing depth > or = 4 mm, with significant reduction post-treatment. Gelatinase activity followed by zymography consisted mainly of MMP-9. A different pattern of MMP-8 in control and patient sites was found. Controls only showed species of a partially active form (69 kDa), whereas patient sites showed a high frequency of the active form (56 kDa), and in some cases the latent form (85 kDa) was also observed. The active form reduced its frequency in sites with probing depth > or = 4 mm. TIMP-1 and -2 levels in patients were significantly lower than in controls, and after treatment the recovery of TIMP-1 level similar to control was observed. Significant correlations between the severity of the periodontal disease and the actual MMP activity, the active form of MMP-8 and the low level of both TIMP-1 and TIMP-2 were found.

Mechanism of Intramembrane Cleavage of Alcadeins by γ-Secretase

PubMed Central

Piao, Yi; Kimura, Ayano; Urano, Satomi; Saito, Yuhki; Taru, Hidenori; Yamamoto, Tohru; Hata, Saori; Suzuki, Toshiharu

2013-01-01

Background Alcadein proteins (Alcs; Alcα, Alcβand Alcγ) are predominantly expressed in neurons, as is Alzheimer's β-amyloid (Aβ) precursor protein (APP). Both Alcs and APP are cleaved by primary α- or β-secretase to generate membrane-associated C-terminal fragments (CTFs). Alc CTFs are further cleaved by γ-secretase to secrete p3-Alc peptide along with the release of intracellular domain fragment (Alc ICD) from the membrane. In the case of APP, APP CTFβ is initially cleaved at the ε-site to release the intracellular domain fragment (AICD) and consequently the γ-site is determined, by which Aβ generates. The initial ε-site is thought to define the final γ-site position, which determines whether Aβ40/43 or Aβ42 is generated. However, initial intracellular ε-cleavage sites of Alc CTF to generate Alc ICD and the molecular mechanism that final γ-site position is determined remains unclear in Alcs. Methodology Using HEK293 cells expressing Alcs plus presenilin 1 (PS1, a catalytic unit of γ-secretase) and the membrane fractions of these cells, the generation of p3-Alc possessing C-terminal γ-cleavage site and Alc ICD possessing N-terminal ε-cleavage site were analysed with MALDI-TOF/MS. We determined the initial ε-site position of all Alcα, Alcβ and Alcγ, and analyzed the relationship between the initially determined ε-site position and the final γ-cleavage position. Conclusions The initial ε-site position does not always determine the final γ-cleavage position in Alcs, which differed from APP. No additional γ-cleavage sites are generated from artificial/non-physiological positions of ε-cleavage for Alcs, while the artificial ε-cleavage positions can influence in selection of physiological γ-site positions. Because alteration of γ-secretase activity is thought to be a pathogenesis of sporadic Alzheimer's disease, Alcs are useful and sensitive substrate to detect the altered cleavage of substrates by γ-secretase, which may be induced by malfunction of γ-secretase itself or changes of membrane environment for enzymatic reaction. PMID:23658629

Speciation of 210Po and 210Pb in air particulates determined by sequential extraction.

PubMed

Al-Masri, M S; Al-Karfan, K; Khalili, H; Hassan, M

2006-01-01

Speciation of (210)Po and (210)Pb in air particulates of two Syrian phosphate sites with different climate conditions has been studied. The sites are the mines and Tartous port at the Mediterranean Sea. Air filters were collected during September 2000 until February 2002 and extracted chemically using different selective fluids in an attempt to identify the different forms of these two radionuclides. The results have shown that the inorganic and insoluble (210)Po and (210)Pb (attached to silica and soluble in mineral acids) portion was found to be high in both sites and reached a maximum value of 94% and 77% in the mine site and Tartous port site, respectively. In addition, only 24% of (210)Pb in air particulates was found to be associated with organic materials probably produced from the incomplete burning of fuel vehicle and similar activities. Moreover, the (210)Po/(210)Pb activity ratio in air particulates was higher than that in all samples at both sites and varied between 3.85 in November 2000 at Tartous port site and 20 in April 2001 at the mine area. These activity ratios were also higher than the natural levels. The (210)Po/(210)Pb activity ratio was also determined in each portion resulting from the selective extraction and found to be higher than that in most samples. The sources of (210)Po excess in these portions are discussed. Soil suspension, which is common in the dry climate dominant in the area, sea water spray and heating of phosphate ores were considered; polonium is more volatile than the lead compounds at even moderate temperature. Furthermore, variations in the chemical forms of (210)Po and (210)Pb during the year were also investigated. However, the results of this study can also be utilized for dose assessment to phosphate industry workers.

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewis-Ballester, Ariel; Pham, Khoa N.; Batabyal, Dipanwita

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination ofmore » an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.« less

Identifying removable radioactivity on the surface of cats during the first week after treatment with iodine 131.

PubMed

Chalmers, H J; Scrivani, Peter V; Dykes, Nathan L; Erb, Hollis N; Hobbs, J M; Hubble, Lorna J

2006-01-01

Because radioiodine (1-131) is excreted in urine and saliva, treated cats can accumulate I-131 on their coats from contacting soiled litter and grooming. This could result in removable radioactivity, which is a potential source of human exposure to radiation and specifically to internal contamination. The purpose of this study was to determine if there is removable radioactivity on cats treated with I-131. Daily wipe tests were performed for 7 days at two sites (both flanks, one site; and all four paws, one site) on six hyperthyroid cats treated with I-131. A y counter was used to determine the counts per minute (cpm) of the samples, which were converted to disintegrations per minute (dpm) to estimate activity. The results were compared to the New York State limits of removable activity for a non-controlled area (<1000dpm/100 cm2) to determine if the amount of removable activity was acceptable for a member of the public. The median value of removable activity was 241 dpm (range from 34 to 4184 dpm) for the flanks, and 308 dpm (range from 60 to 1890 dpm) for the paws. The amount of removable radioactivity on the surface of hospitalized cats treated with I-131 during the first week after treatment, occasionally and without obvious pattern, exceeded the New York State limit. Sporadic activity as high as 4148 dpm was found. It is prudent to advise owners to observe routine hygiene when handling cats after discharge to minimize the risk of internal contamination.

[Analysis of the web pages of the intensive care units of Spain].

PubMed

Navarro-Arnedo, J M

2009-01-01

In order to determine the Intensive Care Units (ICU) of Spanish hospitals that had a web site, to analyze the information they offered and to know what information they needed to offer according to a sample of ICU nurses, a cross-sectional observational, descriptive study was carried out between January and September 2008. For each ICU website, an analysis was made on the information available on the unit, its care, teaching and research activity on nursing. Simultaneously, based on a sample of intensive care nurses, the information that should be contained on an ICU website was determined. The results, expressed in absolute numbers and percentage, showed that 66 of the 292 hospitals with ICU (22.6%) had a web site; 50.7% of the sites showed the number of beds, 19.7% the activity report, 11.3% the published articles/studies and followed research lines and 9.9% the organized formation courses. 14 webs (19.7%) displayed images of nurses. However, only 1 (1.4%) offered guides on the actions followed. No web site offered a navigation section for nursing, the E-mail of the chief nursing, the nursing documentation used or if any nursing model of their own was used. It is concluded that only one-fourth of the Spanish hospitals with ICU have a web site; number of beds was the data offered by the most sites, whereas information on care, educational and investigating activities was very reduced and that on nursing was practically omitted on the web pages of intensive care units.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slade, Daniel J.; Fang, Pengfei; Dreyton, Christina J.

Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ionsmore » that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.« less

Electrical utilities model for determining electrical distribution capacity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fritz, R. L.

1997-09-03

In its simplest form, this model was to obtain meaningful data on the current state of the Site`s electrical transmission and distribution assets, and turn this vast collection of data into useful information. The resulting product is an Electrical Utilities Model for Determining Electrical Distribution Capacity which provides: current state of the electrical transmission and distribution systems; critical Hanford Site needs based on outyear planning documents; decision factor model. This model will enable Electrical Utilities management to improve forecasting requirements for service levels, budget, schedule, scope, and staffing, and recommend the best path forward to satisfy customer demands at themore » minimum risk and least cost to the government. A dynamic document, the model will be updated annually to reflect changes in Hanford Site activities.« less

Arsenic and metals in soils in the vicinity of the Imperial Oil Company Superfund Site, Marlboro Township, Monmouth County, New Jersey. Water-resources investigations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barringer, J.L.; Szabo, Z.; Barringer, T.H.

1998-09-01

Concentrations of arsenic exceed the New Jersey State Cleanup Criterion of 20 parts per million in sandy and clay-rich soils of two residential areas in the vicinity of the Imperial Oil Company Superfund site in Marlboro Township, Monmouth County, New Jersey. In order to determine the source of the arsenic and metals in soils in the two residential areas, soil samples were collected from (1) long-term forested areas, to determine background geologic and regional atmospheric inputs of arsenic and metals; (2) former and current orchards, to assess the range of concentrations of arsenic and metals that could be contributed bymore » past use of pesticides; (3) the Imperial Oil Company Superfund site, to characterize the chemical composition of contamination from activities at the site; (4) a wooded area adjacent to the Superfund site, to determine whether arsenic and metals from the Superfund site were evident; and (5) the two residential areas, to compare soil chemistry in these areas with the chemistry of soils from forests, orchards, and the Superfund site. The soil samples were divided by soil horizon and were analyzed for 23 metals and metalloids, total organic carbon, and total sulfur. Additionally, air-flow models were used to determine whether roasting of arsenic at the Imperial Oil Company Superfund site was a possible source of arsenic in the soils.« less

Three-dimensional structure of porcine pancreatic carboxypeptidase B with an acetate ion and two zinc atoms in the active site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akparov, V. Kh., E-mail: valery@akparov.ru; Timofeev, V. I., E-mail: tostars@mail.ru; Maghsoudi, N. N., E-mail: maghsudi@yahoo.com

2017-03-15

Crystals of porcine pancreatic carboxypeptidase B (CPB) were grown by the capillary counter-diffusion method in the presence of polyethylene glycol and zinc acetate. The three-dimensional structure of CPB was determined at 1.40 Å resolution using the X-ray diffraction data set collected from the crystals of the enzyme at the SPring 8 synchrotron facility and was refined to R{sub fact} = 17.19%, R{sub free} = 19.78%. The structure contains five zinc atoms, two of which are present in the active site of the enzyme, and an acetate ion. The arrangement of an additional zinc atom in the active site and themore » acetate ion is different from that reported by Yoshimoto et al.« less

Supplement Analysis for the Wildlife Mitigation Program EIS (DOE/EIS-0246/SA-36)

DOE Office of Scientific and Technical Information (OSTI.GOV)

N /A

2003-10-10

The compliance checklist for this project was originally completed by the Burns Paiute Tribe in 2000, and meets the standards and guidelines for the Wildlife Mitigation Program Environmental Impact Statement (EIS) and Record of Decision (ROD), as well as the Watershed Management Program Environmental Impact Statement (EIS) and Record of Decision (ROD). The Logan Valley Wildlife Mitigation Plan, now being implemented, continues to be consistent with the above mentioned EISs and RODs. Pursuant to its obligations under the Endangered Species Act, BPA has made a determination of whether its proposed project will have any effects on any listed species undermore » the jurisdiction of the United States Fish and Wildlife Service (USFWS). A species list was obtained from USFWS on June 12, 2003, identifying bald eagles, Canada lynx, and bull trout as potentially occurring in the project area. A site assessment was conducted on July 15, 2003 to determine if these species were present and the potential effects of project activities. A ''No Effect'' determination was made for all ESA-listed species. There were no listed species under the jurisdiction of NOAA Fisheries present in the project area. As management activities proceed in the future, BPA will annually re-assess potential effects of planned activities on listed species. The Burns-Paiute Tribe conducted a literature search for historic and archaeological sites on the property on January 11, 1999. No known sites were identified. Further site-specific surveys will be conducted for individual ground disturbing activities. The results of these surveys will be sent to the Oregon State Historic Preservation Office and BPA. BPA will annually summarize and submit a report to the State Historic Preservation Office. On December 29, 1999, Fred Walasavage of BPA completed a Phase I Site Assessment and concluded that the site did not reveal any environmental factors that would pose a significant liability for remedial action or cleanup under the Comprehensive Recovery, Compensation and Liability Act. A public meeting was held when the property was initially acquired where the property acquisition and proposed activities were discussed. Subsequent public involvement was conducted on July 23, 2002 for commenting on the proposed Logan Valley Wildlife Mitigation Plan.« less

Litter chemistry prevails over litter consumers in mediating effects of past steel industry activities on leaf litter decomposition.

PubMed

Lucisine, Pierre; Lecerf, Antoine; Danger, Michaël; Felten, Vincent; Aran, Delphine; Auclerc, Apolline; Gross, Elisabeth M; Huot, Hermine; Morel, Jean-Louis; Muller, Serge; Nahmani, Johanne; Maunoury-Danger, Florence

2015-12-15

Soil pollution has adverse effects on the performance and life history traits of microorganisms, plants, and animals, yet evidence indicates that even the most polluted sites can support structurally-complex and dynamic ecosystems. The present study aims at determining whether and how litter decomposition, one of the most important soil ecological processes leaf, is affected in a highly trace-metal polluted site. We postulated that past steel mill activities resulting in soil pollution and associated changes in soil characteristics would influence the rate of litter decomposition through two non-exclusive pathways: altered litter chemistry and responses of decomposers to lethal and sub-lethal toxic stress. We carried out a litter-bag experiment using Populus tremula L. leaf litter collected at, and allowed to decompose in, a trace metal polluted site and in three unpolluted sites used as controls. We designed a fully-factorial transplant experimental design to assess effects of litter origin and exposure site on the rate of litter decomposition. We further determined initial litter chemistry, fungal biomass, mesofauna abundance in litter bags, and the soil macrofauna community. Irrespective of the site of litter exposure, litter originating from the polluted site had a two-fold faster decomposition than litter from the unpolluted sites. Litter chemistry, notably the lignin content, seemed most important in explaining the degradation rate of the leaf litter. Abundance of meso and macro-detritivores was higher at the polluted site than at the unpolluted sites. However, litter decomposition proceeded at similar rates in polluted and unpolluted sites. Our results show that trace metal pollution and associated soil and litter changes do not necessarily weaken consumer control on litter decomposition through lethal and sub-lethal toxic stress. Copyright © 2015. Published by Elsevier B.V.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oliver, S. A.; Harris, V. G.; Hamdeh, H. H.

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 zincmore » 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.« less

Site occupation of indium and jump frequencies of cadmium in FeGa 3

NASA Astrophysics Data System (ADS)

Newhouse, Randal; Collins, Gary S.; Zacate, Matthew O.

2016-12-01

Perturbed angular correlation (PAC) measurements using the In-111 probe were carried out on FeGa3 as part of a broader investigation of indium site occupation and cadmium diffusion in intermetallic compounds. One PAC signal was observed with hyperfine parameters ω 1= 513.8(1) Mrad/s and η= 0.939(2) at room temperature. By comparison with quadrupole frequencies observed in PAC measurements on isostructural RuIn3, it was determined that indium occupies only the 8j site in the FeGa3 structure, denoted Ga(2) below because two out of the three Ga sites have this point symmetry. PAC spectra at elevated temperature exhibited damping characteristic of electric field gradients (EFGs) that fluctuate as Cd probes jump among Ga(2) sites within the lifetime of the excited PAC level. A stochastic model for the EFG fluctuations based on four conceivable, single-step jump-pathways connecting one Ga(2) site to neighboring Ga(2) sites was developed and used to fit PAC spectra. The four pathways lead to two observable EFG reorientation rates, and these reorientation rates were found to be strongly dependent on EFG orientation. Calculations using density functional theory were used to reduce the number of unknowns in the model with respect to EFG orientation. This made it possible to determine with reasonable precision the total jump rate of Cd among Ga(2) sites that correspond to a change in mirror plane orientation of site-symmetry. This total jump rate was found to be thermally activated with an activation enthalpy of 1.8 ±0.1 eV.

A Quantitative Approach for Collocating NEON's Sensor-Based Ecological Measurements and in-situ Field Sampling and Observations

NASA Astrophysics Data System (ADS)

Zulueta, R. C.; Metzger, S.; Ayres, E.; Luo, H.; Meier, C. L.; Barnett, D.; Sanclements, M.; Elmendorf, S.

2013-12-01

The National Ecological Observatory Network (NEON) is a continental-scale research platform currently in development to assess the causes of ecological change and biological responses to change across a projected 30-year timeframe. A suite of standardized sensor-based measurements (i.e., Terrestrial Instrument System (TIS) measurements) and in-situ field sampling and observations (i.e., Terrestrial Observation System (TOS) activities) will be conducted across 20 ecoclimatic domains in the U.S. where NEON is establishing 60 terrestrial research sites. NEON's TIS measurements and TOS activities are designed to observe the temporal and spatial dynamics of key drivers and ecological processes and responses to change within each of the 60 terrestrial research sites. The TIS measurements are non-destructive and designed to provide in-situ, continuous, and areally integrated observations of the surrounding ecosystem and environment, while TOS sampling and observation activities are designed to encompass a hierarchy of measurable biological states and processes including diversity, abundance, phenology, demography, infectious disease prevalence, ecohydrology, and biogeochemistry. To establish valid relationships between these drivers and site-specific responses, two contradicting requirements must be fulfilled: (i) both types of observations shall be representative of the same ecosystem, and (ii) they shall not significantly influence one another. Here we outline the theoretical background and algorithmic process for determining areas of mutual representativeness and exclusion around NEON's TIS measurements and develop a procedure which quantitatively optimizes this trade-off through: (i) quantifying the source area distributions of TIS measurements, (ii) determining the ratio of user-defined impact threshold to effective impact area for different TOS activities, and (iii) determining the range of feasible distances between TIS locations and TOS activities. This approach provides an evidence-based and repeatable method for combining sensor-based measurements and field sampling and observations at predefined levels of disturbance and spatial representativeness. The developed approach represents a general framework which is applicable to other environmental research sites where similar collocation is desired.

Evaluating differences in the active-site electronics of supported Au nanoparticle catalysts using Hammett and DFT studies

NASA Astrophysics Data System (ADS)

Kumar, Gaurav; Tibbitts, Luke; Newell, Jaclyn; Panthi, Basu; Mukhopadhyay, Ahana; Rioux, Robert M.; Pursell, Christopher J.; Janik, Michael; Chandler, Bert D.

2018-03-01

Supported metal catalysts, which are composed of metal nanoparticles dispersed on metal oxides or other high-surface-area materials, are ubiquitous in industrially catalysed reactions. Identifying and characterizing the catalytic active sites on these materials still remains a substantial challenge, even though it is required to guide rational design of practical heterogeneous catalysts. Metal-support interactions have an enormous impact on the chemistry of the catalytic active site and can determine the optimum support for a reaction; however, few direct probes of these interactions are available. Here we show how benzyl alcohol oxidation Hammett studies can be used to characterize differences in the catalytic activity of Au nanoparticles hosted on various metal-oxide supports. We combine reactivity analysis with density functional theory calculations to demonstrate that the slope of experimental Hammett plots is affected by electron donation from the underlying oxide support to the Au particles.

Nevada National Security Site Industrial Sites Project Closeout - 12498

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cabble, Kevin; Krauss, Mark; Matthews, Pat

The U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office is responsible for environmental restoration (ER) at the Nevada National Security Site (NNSS). This includes remediation at Industrial Sites where past nuclear testing activities and activities that supported nuclear testing may have or are known to have resulted in the release of contaminants into the environment. Industrial Sites at the NNSS have included nuclear facilities that supported the nuclear rocket/missile development programs, gas stations, landfills, spill sites, ordnance sites, and numerous other waste disposal and release sites. The NNSS Industrial Sites activities neared completion at the endmore » of fiscal year 2011 while other activities required under the Federal Facility Agreement and Consent Order (FFACO) and part of the same NNSS ER Project are forecasted to extend to 2027 or beyond. With the majority of Industrial Sites corrective action units (CAUs) completed (more than 250 CAUs and over 1,800 corrective action sites), it was determined that an activity closeout process should be implemented to ensure that the work completed over the past 15 years is well documented in a comprehensive and concise summary. While the process used to close each individual CAU is described in approved documents, no single document describes in summary fashion the work completed to close the many individual Industrial Sites. The activity closeout process will be used to develop an Industrial Sites closeout document that describes these years of work. This document will summarize the number of Industrial Sites closed under the FFACO and provide general descriptions of projects, contaminants removed, and sites closed in place with corresponding Use Restrictions. Other pertinent information related to Industrial Sites work such as the project history, closure decisions, historical declarations, remediation strategies, and final CAU status will be included in the closeout document, along with a table listing each CAU and corresponding corrective action sites within each CAU. Using this process of conducting the activity closeout and developing a closeout document may prove useful for other ER projects within the DOE complex in describing how a long period of ER can be summarized in a single document. The NNSS Industrial Sites activities were completed over the span of 15 years and involved the investigation, cleanup or Use Restriction, and closure of more than 260 CAUs and over 1,800 sites. These activities will conclude in FY 2012 (with the exception of one CAU). In order to capture the work completed over this length of time and document decisions made during the activities, a closeout effort was initiated. The closeout will review the work conducted during the Industrial Sites activities and produce a single document that summarizes Industrial Sites activities. This closeout is being conducted at an interim stage in the overall NNSA/NSO ER Project since the Soils and UGTA activities will continue for a number of years, but the completion of the Industrial Sites project warrants conducting a closeout now while personnel are available and information is still current. The process followed by NNSA/NSO in conducing project closeout for the Industrial Sites portion of the ER program may prove useful within the DOE complex in demonstrating how a large ER project can be summarized. (authors)« less

Coordination Environment of a Site-Bound Metal Ion in the Hammerhead Ribozyme Determined by 15N and 2H ESEEM Spectroscopy

PubMed Central

Vogt, Matthew; Lahiri, Simanti; Hoogstraten, Charles G.; Britt, R. David; DeRose, Victoria J.

2010-01-01

Although site-bound Mg2+ ions have been proposed to influence RNA structure and function, establishing the molecular properties of such sites has been challenging due largely to the unique electrostatic properties of the RNA biopolymer. We have previously determined that, in solution, the hammerhead ribozyme (a self-cleaving RNA) has a high-affinity metal ion binding site characterized by a Kd,app < 10 µM for Mn2+ in 1 M NaCl and speculated that this site has functional importance in the ribozyme cleavage reaction. Here we determine both the precise location and the hydration level of Mn2+ in this site using ESEEM (electron spin–echo envelope modulation) spectroscopy. Definitive assignment of the high-affinity site to the activity-sensitive A9/G10.1 region is achieved by site-specific labeling of G10.1 with 15N guanine. The coordinated metal ion retains four water ligands as measured by 2H ESEEM spectroscopy. The results presented here show that a functionally important, specific metal binding site is uniquely populated in the hammerhead ribozyme even in a background of high ionic strength. Although it has a relatively high thermodynamic affinity, this ion remains partially hydrated and is chelated to the RNA by just two ligands. PMID:17177426

Structure of the endonuclease IV homologue from Thermotoga maritima in the presence of active-site divalent metal ions

PubMed Central

Tomanicek, Stephen J.; Hughes, Ronny C.; Ng, Joseph D.; Coates, Leighton

2010-01-01

The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5′-phosphodiester bond at an AP site to generate a free 3′-­hydroxyl group and a 5′-terminal sugar phosphate using their AP nuclease activity. Specifically, Thermotoga maritima endonuclease IV is a member of the second conserved AP endonuclease family that includes Escherichia coli endonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-­ray crystal structures of the T. maritima endonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of the T. maritima endonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily. PMID:20823514

Dioxin effects on wood duck (Aix sponsa) embryos from sites near paper mills

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beeman, D.K.; Melancon, M.J.; Fleming, W.J.

Biological and biochemical variables were studied in wood duck embryos from four dioxin-contaminated sites near paper mills in the Southeastern United States and three reference sites. Sites were selected based on a history of dioxin contamination in both sediments and fish. In addition, wood duck embryos collected downstream from an Arkansas Superfund site with demonstrated dioxin-induced reproductive impairment served as positive controls. Whole clutches of eggs were collected from the wild after fifteen days of incubation and mechanically incubated. Two embryos per clutch were sacrificed at pipping and liver monooxygenase activities (BROD, EROD and MROD) were quantified. Hatching success wasmore » determined for the remainder of the nest. Preliminary results indicate no difference in monooxygenase activities across sites even though the authors have previously demonstrated induction of monooxygenase activity in wood duck embryos in laboratory studies. In addition, there were no differences in weight at pipping, liver weight and liver weight to body weight ratios. No differences were seen in hatching success or weight at hatch nor were there any gross morphological abnormalities. This may indicate that exposure of wood ducks nesting near these pulp paper mills is below those which cause elevated monooxygenase activities and reproductive impairment.« less

Inhibition of dog and human gastric lipases by enantiomeric phosphonate inhibitors: a structure-activity study.

PubMed

Miled, Nabil; Roussel, Alain; Bussetta, Cécile; Berti-Dupuis, Liliane; Rivière, Mireille; Buono, Gérard; Verger, Robert; Cambillau, Christian; Canaan, Stéphane

2003-10-14

The crystal structures of gastric lipases in the apo form [Roussel, A., et al. (1999) J. Biol. Chem. 274, 16995-17002] or in complex with the (R(P))-undecyl butyl phosphonate [C(11)Y(4)(+)] [Roussel, A., et al. (2002) J. Biol. Chem. 277, 2266-2274] have improved our understanding of the structure-activity relationships of acid lipases. In this report, we have performed a kinetic study with dog and human gastric lipases (DGL and HGL, respectively) using several phosphonate inhibitors by varying the absolute configuration of the phosphorus atom and the chain length of the alkyl/alkoxy substituents. Using the two previously determined structures and that of a new crystal structure obtained with the other (S(P))-phosphonate enantiomer [C(11)Y(4)(-)], we constructed models of phosphonate inhibitors fitting into the active site crevices of DGL and HGL. All inhibitors with a chain length of fewer than 12 carbon atoms were found to be completely buried in the catalytic crevice, whereas longer alkyl/alkoxy chains were found to point out of the cavity. The main stereospecific determinant explaining the stronger inhibition of the S(P) enantiomers is the presence of a hydrogen bond involving the catalytic histidine as found in the DGL-C(11)Y(4)(-) complex. On the basis of these results, we have built a model of the first tetrahedral intermediate corresponding to the tristearoyl-lipase complex. The triglyceride molecule completely fills the active site crevice of DGL, in contrast with what is observed with other lipases such as pancreatic lipases which have a shallower and narrower active site. For substrate hydrolysis, the supply of water molecules to the active site might be achieved through a lateral channel identified in the protein core.

Crystal structure of KPC-2

PubMed Central

Ke, Wei; Bethel, Christopher R.; Thomson, Jodi M.; Bonomo, Robert A.; van den Akker, Focco

2008-01-01

β-lactamases inactivate β-lactam antibiotics and are a major cause of antibiotic resistance. The recent outbreaks of Klebsiella pneumoniae carbapenem-resistant (KPC) infections mediated by KPC type β-lactamases are creating a serious threat to our “last resort” antibiotics, the carbapenems. KPC β-lactamases are thus carbapenemases and are a subclass of Class A β-lactamases that have evolved to efficiently hydrolyze carbapenems and cephamycins which contain substitutions at the α position proximal to the carbonyl group that normally render these β-lactams resistant to hydrolysis. To investigate the molecular basis of this carbapenemase activity, we have determined the structure of KPC-2 at 1.85Å resolution. The active site of KPC-2 reveals the presence of a bicine buffer molecule which interacts via its carboxyl group with conserved active site residues S130, K234, T235, and T237; this likely resembles the interactions the β-lactam carboxyl moiety makes in the Michaelis-Menten complex. Comparison of the KPC-2 structure with non-carbapenemases and previously determined NMC-A and SME-1 carbapenemase structures shows several active site alterations that are unique among carbapenemases. An outward shift of the catalytic S70 residue renders the active sites of the carbapenemases more shallow likely allowing easier access of the bulkier substrates. Further space for the α-substituents is likely provided by shifts in N132 and N170 in addition to concerted movements in the postulated carboxyl binding pocket that might allow the substrates to bind in a slightly different angle to accommodate these α-substituents. The structure of KPC-2 thus provides key insights into the carbapenemase activity of emerging Class A β-lactamases. PMID:17441734

10 CFR 810.7 - Generally authorized activities.

Code of Federal Regulations, 2011 CFR

2011-01-01

... determined that the following activities are generally authorized, provided no sensitive nuclear technology... continuing programs, to enhance the operational safety of an existing civilian nuclear power plant in a... off-site population posed by a civilian nuclear power plant in such a country; provided the Department...

10 CFR 810.7 - Generally authorized activities.

Code of Federal Regulations, 2010 CFR

2010-01-01

... determined that the following activities are generally authorized, provided no sensitive nuclear technology... continuing programs, to enhance the operational safety of an existing civilian nuclear power plant in a... off-site population posed by a civilian nuclear power plant in such a country; provided the Department...

Modulation of individual steps in group I intron catalysis by a peripheral metal ion.

PubMed

Forconi, Marcello; Piccirilli, Joseph A; Herschlag, Daniel

2007-10-01

Enzymes are complex macromolecules that catalyze chemical reactions at their active sites. Important information about catalytic interactions is commonly gathered by perturbation or mutation of active site residues that directly contact substrates. However, active sites are engaged in intricate networks of interactions within the overall structure of the macromolecule, and there is a growing body of evidence about the importance of peripheral interactions in the precise structural organization of the active site. Here, we use functional studies, in conjunction with published structural information, to determine the effect of perturbation of a peripheral metal ion binding site on catalysis in a well-characterized catalytic RNA, the Tetrahymena thermophila group I ribozyme. We perturbed the metal ion binding site by site-specifically introducing a phosphorothioate substitution in the ribozyme's backbone, replacing the native ligands (the pro-R (P) oxygen atoms at positions 307 and 308) with sulfur atoms. Our data reveal that these perturbations affect several reaction steps, including the chemical step, despite the absence of direct contacts of this metal ion with the atoms involved in the chemical transformation. As structural probing with hydroxyl radicals did not reveal significant change in the three-dimensional structure upon phosphorothioate substitution, the effects are likely transmitted through local, rather subtle conformational rearrangements. Addition of Cd(2+), a thiophilic metal ion, rescues some reaction steps but has deleterious effects on other steps. These results suggest that native interactions in the active site may have been aligned by the naturally occurring peripheral residues and interactions to optimize the overall catalytic cycle.

77 FR 58590 - Determining Technical Adequacy of Probabilistic Risk Assessment for Risk-Informed License...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-09-21

... reactors or for activities associated with review of applications for early site permits and combined licenses (COL) for the Office of New Reactors (NRO). DATES: The effective date of this SRP update is... Rulemaking Web Site: Go to http://www.regulations.gov and search for Docket ID NRC-2010-0138. Address...

Age structure and age-related performance of sulfur cinquefoil (Potentilla recta).

Treesearch

Dana L. Perkins; Catherine G. Parks; Kathleen A. Dwire; Bryan A. Endress; Kelsi L. Johnson

2006-01-01

Age distributions of sulfur cinquefoil populations were determined on sites that were historically grazed, cultivated, and mechanically disturbed. From 12 sites, a total of 279 reproductively active plants were collected and aged by using herbchronology (counting rings in the secondary root xylem of the root crown) to (1) estimate the age structure of the populations...

Commissions as information organizations: Meeting the information needs of an electronic society

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sevel, F.

1997-11-01

This paper describes how commission-sponsored web sites can effectively meet electronic information needs. Demographics of internet users are presented and analyzed. Online activities and user access data are also described. The implications of the characteristics of internet users for commission-sponsored web sites are discussed, and guidelines for determining marketing objectives are presented.

U.S. Nuclear Regulatory Commission Role and Activities Related to U.S. Department of Energy Incidental Waste Determinations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradford, A.H.; Esh, D.W.; Ridge, A.C.

2006-07-01

Section 3116 of the Ronald W. Reagan National Defense Authorization Act for Fiscal Year 2005 (NDAA) requires the U.S. Department of Energy (DOE) to consult with the U.S. Nuclear Regulatory Commission (NRC) for certain non-high level waste (HLW) determinations. Under the NDAA, NRC performs consultative technical reviews of DOE's waste determinations and monitors DOE's disposal actions for such waste, but the NRC does not have regulatory authority over DOE's waste disposal activities. The NDAA provides the criteria that must be met to determine that waste is not HLW. The criteria require that the waste does not need to be disposedmore » of in a geologic repository, that highly radioactive radionuclides be removed to the maximum extent practical, and that the performance objectives of 10 CFR 61, Subpart C, be met. The performance objectives contain criteria for protection of the public, protection of inadvertent intruders, protection of workers, and stability of the disposal site after closure. This paper describes NRC's approach to implementing its responsibilities under the NDAA, as well as similar activities being performed for sites not covered by the NDAA. (authors)« less

A simplified method for active-site titration of lipases immobilised on hydrophobic supports.

PubMed

Nalder, Tim D; Kurtovic, Ivan; Barrow, Colin J; Marshall, Susan N

2018-06-01

The aim of this work was to develop a simple and accurate protocol to measure the functional active site concentration of lipases immobilised on highly hydrophobic supports. We used the potent lipase inhibitor methyl 4-methylumbelliferyl hexylphosphonate to titrate the active sites of Candida rugosa lipase (CrL) bound to three highly hydrophobic supports: octadecyl methacrylate (C18), divinylbenzene crosslinked methacrylate (DVB) and styrene. The method uses correction curves to take into account the binding of the fluorophore (4-methylumbelliferone, 4-MU) by the support materials. We showed that the uptake of the detection agent by the three supports is not linear relative to the weight of the resin, and that the uptake occurs in an equilibrium that is independent of the total fluorophore concentration. Furthermore, the percentage of bound fluorophore varied among the supports, with 50 mg of C18 and styrene resins binding approximately 64 and 94%, respectively. When the uptake of 4-MU was calculated and corrected for, the total 4-MU released via inhibition (i.e. the concentration of functional lipase active sites) could be determined via a linear relationship between immobilised lipase weight and total inhibition. It was found that the functional active site concentration of immobilised CrL varied greatly among different hydrophobic supports, with 56% for C18, compared with 14% for DVB. The described method is a simple and robust approach to measuring functional active site concentration in immobilised lipase samples. Copyright © 2018 Elsevier Inc. All rights reserved.

Impact assessment of a wastewater treatment plant effluent using the fish biomarker ethoxyresorufin-O-deethylase: field and on-site experiments.

PubMed

Kosmala, A; Migeon, B; Flammarion, P; Garric, J

1998-09-01

The impact of a wastewater treatment plant (WWTP) effluent was assessed with the fish biomarker ethoxyresorufin-O-deethylase (EROD) using field and on-site laboratory experiments. EROD activity was measured in chub (Leuciscus cephalus) and stone loach (Noemacheilus barbatulus) caught at three sites of the Chalaronne River (southeast France). Liver somatic index (LSI) and organochloride bioaccumulation in muscle were estimated for chub only. In September, EROD activity and LSI of chub increased significantly between the sites above and below the WWTP effluent discharge. EROD induction detected in chub was confirmed by on-site tank experiments. EROD levels were determined in juvenile rainbow trout (Oncorhynchus mykiss) and mirror carp (Cyprinus carpio) exposed to different concentrations of the WWTP effluent and river water for 16 days. After a 4-day exposure, EROD activities of the carp exposed to the effluent increased significantly compared with the control. The response was linked to the effluent concentration and was stable with exposure time. WWTP effluent induced EROD activity, whereas organic and metal analyses, performed on fish muscle and sediment, did not indicate any difference between upstream and downstream of the discharge. Copyright 1998 Academic Press.

High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase.

PubMed

Romi, Erez; Baran, Nava; Gantman, Marina; Shmoish, Michael; Min, Bosun; Collins, Kathleen; Manor, Haim

2007-05-22

Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in functional enzyme complexes. Our assays reveal contact of the single-stranded DNA adjacent to the primer-template hybrid and tTERT residue W187 at the periphery of the N-terminal domain. This contact was detected in complexes with three different registers of template in the active site, suggesting that it is maintained throughout synthesis of a complete telomeric repeat. Substitution of nearby residue Q168, but not W187, alters the K(m) for primer elongation, implying that it plays a role in the DNA recognition. These findings are the first to directly demonstrate the physical location of TERT-DNA contacts in catalytically active telomerase and to identify amino acid determinants of DNA binding affinity. Our data also suggest a movement of the TERT active site relative to the template-adjacent single-stranded DNA binding site within a cycle of repeat synthesis.

NRC Perspectives on Waste Incidental to Reprocessing Consultations and Monitoring - 13398

DOE Office of Scientific and Technical Information (OSTI.GOV)

McKenney, Christepher A.; Suber, Gregory F.; Felsher, Harry D.

2013-07-01

Section 3116 of the Ronald W. Reagan National Defense Authorization Act for Fiscal Year 2005 (NDAA) requires the U.S. Department of Energy (DOE) to consult with the U.S. Nuclear Regulatory Commission (NRC) for certain non-high level waste (HLW) determinations. The NDAA also requires NRC to monitor DOE's disposal actions related to those determinations to assess compliance with NRC regulations in 10 CFR Part 61, Subpart C. The NDAA applies to DOE activities that will remain within the States of South Carolina and Idaho. DOE has chosen to, under DOE Order 435.1, engage in consultation with NRC for similar activities inmore » the State of Washington and New York, however, the NRC has no monitoring responsibilities. In 2007, the NRC developed a draft Final Report for Interim Use entitled, NUREG-1854: NRC Staff Guidance for Activities Related to U.S. Department of Energy Waste Determinations. Since the law was enacted, the DOE and NRC have consulted on three waste determinations within the affected States: (1) the Saltstone Disposal Facility at the Savannah River Site (SRS) within the State of South Carolina in 2005, (2) the INTEC Tank Farm at the Idaho National Laboratory within the State of Idaho in 2006, and (3) the F Tank Farm at SRS in 2011. After the end of consultation and issuance by DOE of the final waste determination, monitoring began at each of these sites, including the development of monitoring plans. In addition to the NDAA sites, DOE has requested NRC consultation support on both individual tanks and the entire C Tank Farm at the Hanford Nuclear Reservation in the State of Washington. DOE also requested consultation of waste determinations performed on the melter and related feed tanks at the West Valley site in New York that would be disposed offsite. In the next few years, NRC and DOE will consult on the last of the NDAA waste determinations for a while, the H Tank Farm waste determination at SRS. DOE may identify other activities in the future but largely NRC's role will change from doing both consultation and monitoring to being focused on monitoring activities within NDAA. DOE has identified other activities at the Hanford Nuclear Reservation that would continue consultation activities but outside of the NDAA in the future. During the past seven years of consultations and monitoring a number of lessons learned about the process, communication issues, and technical guidance have been identified. With the change in focus from reviewing initial performance assessments and draft waste determinations to long-term monitoring (e.g., individual waste tank closure, at F Tank Farm or complete tank farm closure at INTEC expected in the near future), the NRC is going to revise and update its guidance over the next few years to reflect the lessons learned and the change in focus. In addition to the lessons learned, improvements in the guidance will have to account possible rule and guidance changes underway within Part 61. This paper will discuss the initial plans, approaches, and time lines to revise the guidance within NUREG-1854, including opportunities for public involvement. (authors)« less

Recognition of Lys48-Linked Di-ubiquitin and Deubiquitinating Activities of the SARS Coronavirus Papain-like Protease.

PubMed

Békés, Miklós; van der Heden van Noort, Gerbrand J; Ekkebus, Reggy; Ovaa, Huib; Huang, Tony T; Lima, Christopher D

2016-05-19

Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUb(Lys48) products. To elucidate this specificity, we report the 2.85 Å crystal structure of SARS PLpro bound to a diUb(Lys48) activity-based probe. SARS PLpro binds diUb(Lys48) in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUb(Lys48) chains is predicated on contacts in the S2 site and enhanced by an S1-S1' preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUb(Lys48) specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections. Copyright © 2016 Elsevier Inc. All rights reserved.

Structural and Kinetic Basis for Substrate Selectivity in Populus tremuloides Sinapyl Alcohol Dehydrogenase

PubMed Central

Bomati, Erin K.; Noel, Joseph P.

2005-01-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities. PMID:15829607

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

PubMed

Bomati, Erin K; Noel, Joseph P

2005-05-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

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.

Comparison of topological clustering within protein networks using edge metrics that evaluate full sequence, full structure, and active site microenvironment similarity.

PubMed

Leuthaeuser, Janelle B; Knutson, Stacy T; Kumar, Kiran; Babbitt, Patricia C; Fetrow, Jacquelyn S

2015-09-01

The development of accurate protein function annotation methods has emerged as a major unsolved biological problem. Protein similarity networks, one approach to function annotation via annotation transfer, group proteins into similarity-based clusters. An underlying assumption is that the edge metric used to identify such clusters correlates with functional information. In this contribution, this assumption is evaluated by observing topologies in similarity networks using three different edge metrics: sequence (BLAST), structure (TM-Align), and active site similarity (active site profiling, implemented in DASP). Network topologies for four well-studied protein superfamilies (enolase, peroxiredoxin (Prx), glutathione transferase (GST), and crotonase) were compared with curated functional hierarchies and structure. As expected, network topology differs, depending on edge metric; comparison of topologies provides valuable information on structure/function relationships. Subnetworks based on active site similarity correlate with known functional hierarchies at a single edge threshold more often than sequence- or structure-based networks. Sequence- and structure-based networks are useful for identifying sequence and domain similarities and differences; therefore, it is important to consider the clustering goal before deciding appropriate edge metric. Further, conserved active site residues identified in enolase and GST active site subnetworks correspond with published functionally important residues. Extension of this analysis yields predictions of functionally determinant residues for GST subgroups. These results support the hypothesis that active site similarity-based networks reveal clusters that share functional details and lay the foundation for capturing functionally relevant hierarchies using an approach that is both automatable and can deliver greater precision in function annotation than current similarity-based methods. © 2015 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

PubMed Central

Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

2016-01-01

Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332

Comparison of topological clustering within protein networks using edge metrics that evaluate full sequence, full structure, and active site microenvironment similarity

PubMed Central

Leuthaeuser, Janelle B; Knutson, Stacy T; Kumar, Kiran; Babbitt, Patricia C; Fetrow, Jacquelyn S

2015-01-01

The development of accurate protein function annotation methods has emerged as a major unsolved biological problem. Protein similarity networks, one approach to function annotation via annotation transfer, group proteins into similarity-based clusters. An underlying assumption is that the edge metric used to identify such clusters correlates with functional information. In this contribution, this assumption is evaluated by observing topologies in similarity networks using three different edge metrics: sequence (BLAST), structure (TM-Align), and active site similarity (active site profiling, implemented in DASP). Network topologies for four well-studied protein superfamilies (enolase, peroxiredoxin (Prx), glutathione transferase (GST), and crotonase) were compared with curated functional hierarchies and structure. As expected, network topology differs, depending on edge metric; comparison of topologies provides valuable information on structure/function relationships. Subnetworks based on active site similarity correlate with known functional hierarchies at a single edge threshold more often than sequence- or structure-based networks. Sequence- and structure-based networks are useful for identifying sequence and domain similarities and differences; therefore, it is important to consider the clustering goal before deciding appropriate edge metric. Further, conserved active site residues identified in enolase and GST active site subnetworks correspond with published functionally important residues. Extension of this analysis yields predictions of functionally determinant residues for GST subgroups. These results support the hypothesis that active site similarity-based networks reveal clusters that share functional details and lay the foundation for capturing functionally relevant hierarchies using an approach that is both automatable and can deliver greater precision in function annotation than current similarity-based methods. PMID:26073648

Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

PubMed

Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

2016-07-19

The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes.

Live, Model, Learn: Experiencing Information Systems Requirements through Simulation

ERIC Educational Resources Information Center

Hartzel, Kathleen S.; Pike, Jacqueline C.

2015-01-01

Information system professionals strive to determine requirements by interviewing clients, observing activities at the client's site, and studying existing system documentation. Still this often leads to vague and inaccurate requirements documentation. When teaching the skills needed to determine requirements, it is important to recreate a…

A study on the determination of the natural park's sustainable tourism potential.

PubMed

Cetin, Mehmet; Zeren, Ilknur; Sevik, Hakan; Cakir, Cansel; Akpinar, Huseyin

2018-02-23

The surface site of Yesilyuva Nature Park encompasses natural, social, economic, and cultural characteristics and has become a marker of the region's natural and cultural heritage. To support the preservation of this site, promotional activities should be planned. In this study, because of tourism and related opinions of residents and visitors alike in terms of their natural determination, an important cultural and historical feature is aimed at evaluating the tourism potential of Yesilyuva Nature Park. This framework is designed to establish prospective tourism sustainability. As a result, Yesilyuva Nature Park's natural and cultural properties have been determined to be suitable for sustainable tourism activities using geographic information systems (GIS). This protection in the field, which balances sustainability and landscape design, will provide for the development of tourism activities. In the Strengths, Weaknesses, Opportunities and Threats (SWOT) analysis and survey, residents and visitors reported that the most important feature of the Yesilyuva Nature Park was its natural beauty. Visitors often come to observe traditional and natural life and to engage in tourism activities. All the data, which includes maps derived from GIS, represents landscape planning for sustainable tourism areas in Yesilyuva Nature Park.

Assessment of potential migration of radionuclides and trace elements from the White Mesa uranium mill to the Ute Mountain Ute Reservation and surrounding areas, southeastern Utah

USGS Publications Warehouse

Naftz, David L.; Ranalli, Anthony J.; Rowland, Ryan C.; Marston, Thomas M.

2011-01-01

In 2007, the Ute Mountain Ute Tribe requested that the U.S. Environmental Protection Agency and U.S. Geological Survey conduct an independent evaluation of potential offsite migration of radionuclides and selected trace elements associated with the ore storage and milling process at an active uranium mill site near White Mesa, Utah. Specific objectives of this study were (1) to determine recharge sources and residence times of groundwater surrounding the mill site, (2) to determine the current concentrations of uranium and associated trace elements in groundwater surrounding the mill site, (3) to differentiate natural and anthropogenic contaminant sources to groundwater resources surrounding the mill site, (4) to assess the solubility and potential for offsite transport of uranium-bearing minerals in groundwater surrounding the mill site, and (5) to use stream sediment and plant material samples from areas surrounding the mill site to identify potential areas of offsite contamination and likely contaminant sources.

Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics

PubMed Central

Doolittle-Hall, Janet M.; Cunningham Glasspoole, Danielle L.; Seaman, William T.; Webster-Cyriaque, Jennifer

2015-01-01

Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV), hepatitis B virus (HBV) or Merkel cell polyomavirus (MCPyV). These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats) and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures. PMID:26569308

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

1997-01-01

An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Oncoprotein protein kinase antibody kit

DOEpatents

Karin, Michael [San Diego, CA; Hibi, Masahiko [San Diego, CA; Lin, Anning [La Jolla, CA

2008-12-23

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

2003-02-04

An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

1997-01-01

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

1998-01-01

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

3’UTR Shortening Potentiates MicroRNA-Based Repression of Pro-differentiation Genes in Proliferating Human Cells

PubMed Central

Hoffman, Yonit; Bublik, Debora Rosa; P. Ugalde, Alejandro; Elkon, Ran; Biniashvili, Tammy; Agami, Reuven; Oren, Moshe; Pilpel, Yitzhak

2016-01-01

Most mammalian genes often feature alternative polyadenylation (APA) sites and hence diverse 3’UTR lengths. Proliferating cells were reported to favor APA sites that result in shorter 3’UTRs. One consequence of such shortening is escape of mRNAs from targeting by microRNAs (miRNAs) whose binding sites are eliminated. Such a mechanism might provide proliferation-related genes with an expression gain during normal or cancerous proliferation. Notably, miRNA sites tend to be more active when located near both ends of the 3’UTR compared to those located more centrally. Accordingly, miRNA sites located near the center of the full 3’UTR might become more active upon 3'UTR shortening. To address this conjecture we performed 3' sequencing to determine the 3' ends of all human UTRs in several cell lines. Remarkably, we found that conserved miRNA binding sites are preferentially enriched immediately upstream to APA sites, and this enrichment is more prominent in pro-differentiation/anti-proliferative genes. Binding sites of the miR17-92 cluster, upregulated in rapidly proliferating cells, are particularly enriched just upstream to APA sites, presumably conferring stronger inhibitory activity upon shortening. Thus 3’UTR shortening appears not only to enable escape from inhibition of growth promoting genes but also to potentiate repression of anti-proliferative genes. PMID:26908102

Metal Dependence of the Xylose Isomerase from Piromyces sp. E2 Explored by Activity Profiling and Protein Crystallography

PubMed Central

2017-01-01

Xylose isomerase from Piromyces sp. E2 (PirXI) can be used to equip Saccharomyces cerevisiae with the capacity to ferment xylose to ethanol. The biochemical properties and structure of the enzyme have not been described even though its metal content, catalytic parameters, and expression level are critical for rapid xylose utilization. We have isolated the enzyme after high-level expression in Escherichia coli, analyzed the metal dependence of its catalytic properties, and determined 12 crystal structures in the presence of different metals, substrates, and substrate analogues. The activity assays revealed that various bivalent metals can activate PirXI for xylose isomerization. Among these metals, Mn2+ is the most favorable for catalytic activity. Furthermore, the enzyme shows the highest affinity for Mn2+, which was established by measuring the activation constants (Kact) for different metals. Metal analysis of the purified enzyme showed that in vivo the enzyme binds a mixture of metals that is determined by metal availability as well as affinity, indicating that the native metal composition can influence activity. The crystal structures show the presence of an active site similar to that of other xylose isomerases, with a d-xylose binding site containing two tryptophans and a catalytic histidine, as well as two metal binding sites that are formed by carboxylate groups of conserved aspartates and glutamates. The binding positions and conformations of the metal-coordinating residues varied slightly for different metals, which is hypothesized to contribute to the observed metal dependence of the isomerase activity. PMID:29045784

Determination of precursor sites for pitting corrosion of polycrystalline titanium by using different techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garfias-Mesias, L.F.; Alodan, M.; James, P.I.

1998-06-01

Scanning electrochemical microscopy (SECM) in ferrocyanide and bromide solutions was used to locate active sites (pitting precursors) on polycrystalline Ti where oxidation of Br{sup {minus}} and Fe(CN){sub 6}{sup 4{minus}} was possible. Analysis of the electrochemically active sites was done by using electron microscopy (SEM), energy dispersive X-ray analysis (EDX), atomic force microscopy (AFM), and in situ confocal laser scanning microscopy (CLSM). In most cases, the active sites were found to be associated with particles (inclusions) which contained mainly Al and Si; however, some other areas not associated with particles were also found to be active. Although the size of themore » inclusions was normally smaller than 20 {micro}m, as revealed by SEM and AFM imaging, in some cases larger particles were also found. Pitting corrosion tests in bromide solution at potentials above 1.5 V{sub SCE} followed by EDX analysis inside the pits and in situ CLSM observation, confirmed that most of the localized attack started in the areas where particles had been located.« less

A comparative theoretical study of the catalytic activities of Au2(-) and AuAg(-) dimers for CO oxidation.

PubMed

Liu, Peng; Song, Ke; Zhang, Dongju; Liu, Chengbu

2012-05-01

The detailed mechanisms of catalytic CO oxidation over Au(2)(-) and AuAg(-) dimers, which represent the simplest models for monometal Au and bimetallic Au-Ag nanoparticles, have been studied by performing density functional theory calculations. It is found that both Au(2)(-) and AuAg(-) dimers catalyze the reaction according to the similar mono-center Eley-Rideal mechanism. The catalytic reaction is of the multi-channel and multi-step characteristic, which can proceed along four possible pathways via two or three elementary steps. In AuAg(-), the Au site is more active than the Ag site, and the calculated energy barrier values for the rate-determining step of the Au-site catalytic reaction are remarkably smaller than those for both the Ag-site catalytic reaction and the Au(2)(-) catalytic reaction. The better catalytic activity of bimetallic AuAg(-) dimer is attributed to the synergistic effect between Au and Ag atom. The present results provide valuable information for understanding the higher catalytic activity of Au-Ag nanoparticles and nanoalloys for low-temperature CO oxidation than either pure metallic catalyst.

Protective effect of extract of Crataegus pinnatifida pollen on DNA damage response to oxidative stress.

PubMed

Cheng, Ni; Wang, Yuan; Gao, Hui; Yuan, Jialing; Feng, Fan; Cao, Wei; Zheng, Jianbin

2013-09-01

The protective effect of extract of Crataegus pinnatifida (Rosaceae) pollen (ECPP) on the DNA damage response to oxidative stress was investigated and assessed with an alkaline single-cell gel electrophoresis (SCGE) assay and pBR322 plasmid DNA breaks in site-specific and non-site-specific systems. Total phenolic content, total flavonoid content, individual phenolic compounds, antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH), radical scavenging activity, FRAP, and chelating activity) were also determined. The results showed that ECPP possessed a strong ability to protect DNA from being damaged by hydroxyl radicals in both the site-specific system and the non-site-specific system. It also exhibited a cytoprotection effect in mouse lymphocytes against H₂O₂-induced DNA damage. These protective effects may be related to its high total phenolic content (17.65±0.97 mg GAE/g), total flavonoid content (8.04±0.97 mg rutin/g), strong free radical scavenging activity and considerable ferrous ion chelating ability (14.48±0.21 mg Na₂EDTA/g). Copyright © 2013 Elsevier Ltd. All rights reserved.

Seafloor massive sulfide deposits support unique megafaunal assemblages: Implications for seabed mining and conservation.

PubMed

Boschen, Rachel E; Rowden, Ashley A; Clark, Malcolm R; Pallentin, Arne; Gardner, Jonathan P A

2016-04-01

Mining of seafloor massive sulfides (SMS) is imminent, but the ecology of assemblages at SMS deposits is poorly known. Proposed conservation strategies include protected areas to preserve biodiversity at risk from mining impacts. Determining site suitability requires biological characterisation of the mine site and protected area(s). Video survey of a proposed mine site and protected area off New Zealand revealed unique megafaunal assemblages at the mine site. Significant relationships were identified between assemblage structure and environmental conditions, including hydrothermal features. Unique assemblages occurred at both active and inactive chimneys and are particularly at risk from mining-related impacts. The occurrence of unique assemblages at the mine site suggests that the proposed protected area is insufficient alone and should instead form part of a network. These results provide support for including hydrothermally active and inactive features within networks of protected areas and emphasise the need for quantitative survey data of proposed sites. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

An integrated assessment of pollution and biological effects in flounder, mussels and sediment in the southern Baltic Sea coastal area.

PubMed

Dabrowska, Henryka; Kopko, Orest; Lehtonen, Kari K; Lang, Thomas; Waszak, Ilona; Balode, Maija; Strode, Evita

2017-02-01

Organic and metal contaminants and biological effects were investigated in flounder, mussels, and sediments in the southern Baltic Sea coastal area in order to assess environmental quality status in that area. Four sites were selected, including two within the Gulf of Gdańsk (GoG). In biota and sediment at each site, DDTs dominated over PCBs and PBDEs were the least abundant among organic contaminants. Their concentrations decreased progressively outward from GoG. Among metal contaminants, the levels of Hg, Pb, and Cd were elevated in GoG. Biomarkers in flounder, EROD activity and DNA SB, showed moderate positive correlations with organic and metal contaminants. In flounder, the integrated biomarker index (IBR/n) presented a spatial trend coherent with chemical pollution index (CPI), but there was no clear spatial correspondence between IBR/n and CPI in mussels nor between sediment toxicity index (STI) and sediment CPI. The integrated assessment of contaminant and biological effect data against available assessment criteria indicated that in biota, the contaminant assessment thresholds were most often exceeded by CB-118, heptachlor, PBDE, and Hg (in the GoG sediments by p,p'-DDT, Hg and Cd), while of the biological determinants, the threshold was breeched by AChE activity in mussels in GoG. Applying the ICES/OSPAR traffic-light approach showed that of the 50 parameters assessed at each site, there were 18% of determinants in the red color category in the two GoG sites and 8% of determinants in the two sites outside GoG, which indicated that none of the four investigated sites attained good environmental status (GES).

Project PARTnership: Instructional Kit. A Model Program for Encouraging Self-Determination through Access to the Arts.

ERIC Educational Resources Information Center

Harris, Carolyn DeMeyer, Ed.; McKinney, David D., Ed.

This instructional kit outlines procedures for implementing Project PARTnership, a program for teaching students with disabilities greater self-determination skills through specifically designed arts experiences. An introductory section describes key project activities, including establishing a site steering committee comprised of teachers,…

Recurrent patterns of atrial depolarization during atrial fibrillation assessed by recurrence plot quantification.

PubMed

Censi, F; Barbaro, V; Bartolini, P; Calcagnini, G; Michelucci, A; Gensini, G F; Cerutti, S

2000-01-01

The aim of this study was to determine the presence of organization of atrial activation processes during atrial fibrillation (AF) by assessing whether the activation sequences are wholly random or are governed by deterministic mechanisms. We performed both linear and nonlinear analyses based on the cross correlation function (CCF) and recurrence plot quantification (RPQ), respectively. Recurrence plots were quantified by three variables: percent recurrence (PR), percent determinism (PD), and entropy of recurrences (ER). We recorded bipolar intra-atrial electrograms in two atrial sites during chronic AF in 19 informed subjects, following two protocols. In one, both recording sites were in the right atrium; in the other protocol, one site was in the right atrium, the other one in the left atrium. We extracted 19 episodes of type I AF (Wells' classification). RPQ detected transient recurrent patterns in all the episodes, while CCF was significant only in ten episodes. Surrogate data analysis, based on a cross-phase randomization procedure, decreased PR, PD, and ER values. The detection of spatiotemporal recurrent patterns together with the surrogate data results indicate that during AF a certain degree of local organization exists, likely caused by deterministic mechanisms of activation.

Potent Mechanism-Based Inactivation Of Cytochrome P450 2B4 By 9-Ethynylphenanthrene: Implications For Allosteric Modulation Of Cytochrome P450 Catalysis1

PubMed Central

Zhang, Haoming; Gay, Sean C.; Shah, Manish; Foroozesh, Maryam; Liu, Jiawang; Osawa, Yoichi; Zhang, Qinghai; Stout, C. David; Halpert, James R.; Hollenberg, Paul F.

2013-01-01

The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and kinact are 0.2 and 0.25 min−1, respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and S50 of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by ESI-LC/MS, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to the Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206 leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low affinity site being the catalytic site and the high affinity site on the protein periphery. Computer-aided docking and MD simulations with one or two ligands bound revealed that the high affinity site is situated at the entrance of a substrate access channel surrounded by the F’ helix, β1/β2 loop and β4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302. PMID:23276288

Estimating the onset of cambial activity in Scots pine in northern Finland by means of the heat-sum approach.

PubMed

Seo, Jeong-Wook; Eckstein, Dieter; Jalkanen, Risto; Rickebusch, Sophie; Schmitt, Uwe

2008-01-01

We estimated the date of onset (Date(est)) of cambial activity by the pinning method in Scots pine (Pinus sylvestris L.) trees at Vanttauskoski (Site 1) and Laanila (Site 2) near the latitudinal limit of Scots pine in northern Finland. In each year and at each site, observations were made on a different set of five trees. The estimated dates of onset of cambial activity were compared with the corresponding heat sums, calculated in degree-days according to two models. Within years, Date(est) varied among trees by up to 15 days at Site 1 and up to 13 days at Site 2. Among years, mean Date(est) varied by 15.3 days at Site 1 and 12.0 days at Site 2. The overall mean Date(est) differed between sites by 6 days (June 5 at Site 1 and June 11 at Site 2). Among all trees in all years, the mean number of degree days (d.d.) calculated from mean daily temperature above a threshold of 5 degrees C before Date(est) ranged from 68.7 to 135 d.d. at Site 1 and from 37.4 to 154.7 d.d. at Site 2. Among years, the mean heat sum before Date(est )ranged from 94 to 112.5 d.d. at Site 1 and from 61.4 to 136 d.d. at Site 2. Variation among years in heat sum before Date(est) at Site 2 was highly significant, indicating that one or more factors other than, or in addition to, heat sum determines the onset of cambial activity in Scots pine. Similar results were obtained when heat sum was computed from the area between the sine wave generated by daily maximum and minimum temperature and the threshold temperature.

Yucca Mountain Biological Resources Monitoring Program. Progress report, January 1994--December 1994

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1995-07-01

The US Department of Energy (DOE) is required by the Nuclear Waste Policy Act of 1982 (as amended in 1987) to study and characterize the suitability of Yucca Mountain as a potential geological repository for high-level nuclear waste. During site characterization, the DOE will conduct a variety of geotechnical, geochemical, geological, and hydrological studies to determine the suitability of Yucca Mountain as a potential repository. To ensure that site characterization activities do not adversely affect the environment at Yucca Mountain, a program has been implemented to monitor and mitigate potential impacts and ensure activities comply with applicable environmental regulations. Thismore » report describes the activities and accomplishments of EG and G Energy Measurements, Inc. (EG and G/EM) from January 1994 through December 1994 for six program areas within the Terrestrial Ecosystem component of the environmental program for the Yucca Mountain Site Characterization Project (YMP): Site Characterization Effects, Desert Tortoises (Gopherus agassizii), Habitat Reclamation, Monitoring and Mitigation, Radiological Monitoring, and Biological Support.« less

Yucca Mountain biological resources monitoring program; Annual report FY92

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1993-02-01

The US Department of Energy (DOE) is required by the Nuclear Waste Policy Act of 1982 (as amended in 1987) to study and characterize Yucca Mountain as a potential site for a geologic repository for high-level nuclear waste. During site characterization, the DOE will conduct a variety of geotechnical, geochemical, geological, and hydrological studies to determine the suitability of Yucca Mountain as a potential repository. To ensure that site characterization activities (SCA) do not adversely affect the environment at Yucca Mountain, an environmental program has been implemented to monitor and mitigate potential impacts and ensure activities comply with applicable environmentalmore » regulations. This report describes the activities and accomplishments of EG&G Energy Measurements, Inc. (EG&G/EM) during fiscal year 1992 (FY92) for six program areas within the Terrestrial Ecosystem component of the YMP environmental program. The six program areas are Site Characterization Effects, Desert Tortoises, Habitat Reclamation, Monitoring and Mitigation, Radiological Monitoring, and Biological Support.« less

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.

The Strength of an Ig Switch Region is Determined by its Ability to Drive R-loop Formation and its Number of WGCW Sites

PubMed Central

Zhang, Zheng Z.; Pannunzio, Nicholas R.; Han, Li; Hsieh, Chih-Lin; Yu, Kefei; Lieber, Michael R.

2014-01-01

SUMMARY R-loops exist at the murine IgH switch regions and possibly other locations, but their functional importance is unclear. In biochemical systems, R-loop initiation requires DNA sequence regions containing clusters of G nucleotides, but cellular studies have not been done. Here, we vary the G-clustering, total switch region length, and the number of target sites (WGCW sites for the activation-induced deaminase) at synthetic switch regions in a murine B cell line to determine the effect on class switch recombination (CSR). G-clusters increase CSR, regardless of their immediate proximity to the WGCW sites. This increase is accompanied by an increase in R-loop formation. CSR efficiency correlates better with the absolute number of WGCW sites in the switch region rather than the total switch region length or density of WGCW sites. Thus, the overall strength of the switch region depends on G-clusters, which initiate R-loop formation, and on the number of WGCW sites. PMID:25017067

PIT-tagged particle study of bed mobility in a Maine salmon river impacted by logging activities

NASA Astrophysics Data System (ADS)

Thompson, D. M.; Fixler, S. A.; Roberts, K. E.; McKenna, M.; Marshall, A. E.; Koenig, S.

2017-12-01

Presenting an interim report on a study on the Narraguagus River in Maine, which utilizes laser total stations cross-sectional surveys and tracking of passive integrated transponder (PIT) tags embedded in glass spheres to document changes in channel-bed characteristics associated with large wood (LW) additions and natural spawning activities. In 2016, work was initiated to monitor changes in bed elevation and sediment mobility with the addition of LW to the Narraguagus River as part of a restoration effort. Ten cross-sections, spaced 5-m apart, were established and surveyed with a laser total station in each of three different study reaches. The study sites include a control reach, a section with anticipated spawning activities and a site with ongoing LW placement. A grid of 200 glass spheres embedded with PIT tags, with twenty alternating 25-mm and 40-mm size particles equally spaced along each of the ten transects, were placed to serve as point sensors to detect sediment mobilization within each reach. In 2017, the site was revisited to determine if differences in PIT-tagged tracer particle mobilization reflect locations were LW was added and places where Atlantic salmon (Salmo salar) and sea lamprey (Petromyzon marinus) construct spawning redds. The positions of PIT-tagged tracer particles was recorded, but particles were not disturbed or uncovered to permit study of potential reworking of buried tracer particles the following year. Full tracer particle recovery will be determined in 2018 to determine if depths of tracer burial and changes in bed elevation vary among places near redds, LW and main channel locations. The data will be used to determine if salmon redds are preferentially located in either places with greater evidence of sediment reworking or alternatively in stable areas? The study will help determine the degree of bed disruption associated with spawning activities and whether LW placement encourages similar sediment mobilization processes.

Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration*

PubMed Central

Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W.

2014-01-01

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions. PMID:24532791

Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration.

PubMed

Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W

2014-03-28

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.

Gene activity test determines cold tolerance in Douglas-fir seedlings

Treesearch

Peter A. Balk; Diane L. Haase; Monique F. van Wordragen

2008-01-01

Forest tree nurseries rely on a tight scheduling of operations to be able to deliver vigorous seedlings to the planting site. Cooler or freezer storage is often used to maintain planting stock in an inactive condition and to ensure a plant supply for geographically diverse planting sites, which is a requirement for large-scale or internationally operating nurseries....

New insights into the catalytic mechanism of Bombyx mori prostaglandin E synthase gained from structure–function analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamamoto, Kohji, E-mail: yamamok@agr.kyushu-u.ac.jp; Suzuki, Mamoru; Higashiura, Akifumi

2013-11-01

Highlights: •Structure of Bombyx mori prostaglandin E synthase is determined. •Bound glutathione sulfonic acid is located at the glutathione-binding site. •Electron-sharing network is present in this protein. •This network includes Asn95, Asp96, and Arg98. •Site-directed mutagenesis reveals that the residues contribute to the catalytic activity. -- Abstract: Prostaglandin E synthase (PGES) catalyzes the isomerization of PGH{sub 2} to PGE{sub 2}. We previously reported the identification and structural characterization of Bombyx mori PGES (bmPGES), which belongs to Sigma-class glutathione transferase. Here, we extend these studies by determining the structure of bmPGES in complex with glutathione sulfonic acid (GTS) at a resolutionmore » of 1.37 Å using X-ray crystallography. GTS localized to the glutathione-binding site. We found that electron-sharing network of bmPGES includes Asn95, Asp96, and Arg98. Site-directed mutagenesis of these residues to create mutant forms of bmPGES mutants indicate that they contribute to catalytic activity. These results are, to our knowledge, the first to reveal the presence of an electron-sharing network in bmPGES.« less

Evaluation of neutron flux parameters in irradiation sites of research reactor using the Westcott-formalism for the k0 neutron activation analysis method

NASA Astrophysics Data System (ADS)

Kasban, H.; Hamid, Ashraf

2015-12-01

Instrumental Neutron Activation Analysis using k0 (k0-INAA) method has been used to determine a number of elements in sediment samples collected from El-Manzala Lake in Egypt. k0-INAA according to Westcott's formalism has been implemented using the complete irradiation kit of the fast pneumatic rabbit and some selected manually loaded irradiation sites for short and long irradiation at Egypt Second Research Reactor (ETRR-2). Zr-Au and Co sets as neutron flux monitors are used to determine the neutron flux parameters (f and α) in each irradiation sites. Two reference materials IAEA Soil-7 samples have been inserted and implemented for data validation and an internal monostandard multi monitor used (k0 based IM-NAA). It was given a good agreement between the experimental analyzed values and that obtained of the certified values. The major and trace elements in the sediment samples have been evaluated with the use of Co as an internal and Au as an external monostandard comparators. The concentrations of the elements (Cr, Mn and Zn) in the sediment samples of the present work are discussed regarding to those obtained from other sites.

10 CFR 765.21 - Procedures for processing reimbursement claims.

Code of Federal Regulations, 2013 CFR

2013-01-01

... Section 765.21 Energy DEPARTMENT OF ENERGY REIMBURSEMENT FOR COSTS OF REMEDIAL ACTION AT ACTIVE URANIUM AND THORIUM PROCESSING SITES Procedures for Submitting and Processing Reimbursement Claims § 765.21... specified in § 765.20(g) to determine the completeness of each claim. Payments from the Fund to active...

Measurement of Aerosolization of Asbestos from Soil Using the Releasable Asbestos Field Sampler (RAFS)

EPA Science Inventory

Releasable Asbestos Field Sampler (RAFS) was designed to provide an alternative to activity-based sampling (ABS) for determining likely asbestos exposure resulting from activities at site with low contaminant levels. This paper presents a description of the RAFS device and a comp...

Facile Site-Directed Mutagenesis of Large Constructs Using Gibson Isothermal DNA Assembly.

PubMed

Yonemoto, Isaac T; Weyman, Philip D

2017-01-01

Site-directed mutagenesis is a commonly used molecular biology technique to manipulate biological sequences, and is especially useful for studying sequence determinants of enzyme function or designing proteins with improved activity. We describe a strategy using Gibson Isothermal DNA Assembly to perform site-directed mutagenesis on large (>~20 kbp) constructs that are outside the effective range of standard techniques such as QuikChange II (Agilent Technologies), but more reliable than traditional cloning using restriction enzymes and ligation.

In vitro toxicity of particulate matter (PM) collected at different sites in the Netherlands is associated with PM composition, size fraction and oxidative potential - the RAPTES project

PubMed Central

2011-01-01

Background Ambient particulate matter (PM) exposure is associated with respiratory and cardiovascular morbidity and mortality. To what extent such effects are different for PM obtained from different sources or locations is still unclear. This study investigated the in vitro toxicity of ambient PM collected at different sites in the Netherlands in relation to PM composition and oxidative potential. Method PM was sampled at eight sites: three traffic sites, an underground train station, as well as a harbor, farm, steelworks, and urban background location. Coarse (2.5-10 μm), fine (< 2.5 μm) and quasi ultrafine PM (qUF; < 0.18 μm) were sampled at each site. Murine macrophages (RAW 264.7 cells) were exposed to increasing concentrations of PM from these sites (6.25-12.5-25-50-100 μg/ml; corresponding to 3.68-58.8 μg/cm2). Following overnight incubation, MTT-reduction activity (a measure of metabolic activity) and the release of pro-inflammatory markers (Tumor Necrosis Factor-alpha, TNF-α; Interleukin-6, IL-6; Macrophage Inflammatory Protein-2, MIP-2) were measured. The oxidative potential and the endotoxin content of each PM sample were determined in a DTT- and LAL-assay respectively. Multiple linear regression was used to assess the relationship between the cellular responses and PM characteristics: concentration, site, size fraction, oxidative potential and endotoxin content. Results Most PM samples induced a concentration-dependent decrease in MTT-reduction activity and an increase in pro-inflammatory markers with the exception of the urban background and stop & go traffic samples. Fine and qUF samples of traffic locations, characterized by a high concentration of elemental and organic carbon, induced the highest pro-inflammatory activity. The pro-inflammatory response to coarse samples was associated with the endotoxin level, which was found to increase dramatically during a three-day sample concentration procedure in the laboratory. The underground samples, characterized by a high content of transition metals, showed the largest decrease in MTT-reduction activity. PM size fraction was not related to MTT-reduction activity, whereas there was a statistically significant difference in pro-inflammatory activity between Fine and qUF PM. Furthermore, there was a statistically significant negative association between PM oxidative potential and MTT-reduction activity. Conclusion The response of RAW264.7 cells to ambient PM was markedly different using samples collected at various sites in the Netherlands that differed in their local PM emission sources. Our results are in support of other investigations showing that the chemical composition as well as oxidative potential are determinants of PM induced toxicity in vitro. PMID:21888644

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.

Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase.

PubMed Central

Allawi, H T; Dong, F; Ip, H S; Neri, B P; Lyamichev, V I

2001-01-01

A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5'-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated ras (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit beta-globin, and human interferon-gamma (IFN-gamma). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility. PMID:11233988

A Finite-Rate-Catalytic Model For Hypersonic Flows Informed By Molecular Dynamics

NASA Astrophysics Data System (ADS)

Schwartzentruber, T. E.; Valentini, P.; Norman, P.; Sorensen, C.

2011-05-01

The implementation of a finite-rate catalytic (FRC) wall boundary condition within a general 3D unstructured CFD solver is described. A set of one-step gas-surface chemical equations and atomistic parameters that deter- mine the reaction rates must be prescribed as input to the model. The chemical rate equations are solved at each wall face in the CFD simulation and result in a net production of species at the wall. In order for a finite- rate gas-surface reaction model to be consistent at equilibrium, it is determined that not all forward and back- ward rates can be specified arbitrarily. Provided that the forward rates for each surface recombination are as- signed, the backward rates must be determined using equilibrium constants that are consistent with the gas- phase chemistry model and thermodynamics. Reactive molecular dynamics (MD) simulations are performed us- ing the ReaxFFSiO potential to investigate oxygen-silica interactions. β-quartz and amorphous SiO2 surfaces are accommodated to a high temperature gas via MD simulation and reach a steady-state surface coverage. In addition to stable surface reconstructions a number of active sites are observed on which recombination occurs. Single collision MD simulations are performed where gas-phase oxygen atoms interact with the most dominant active site. Probabilities of recombination are found to have an exponential trend with gas-surface system temperature. The MD simulations are used to determine the activation energy for Eley-Rideal recombination of oxygen on a specific silica active site which is an important input parameter for the FRC model.

Plant uptake of 238U, 235U, 232Th, 226Ra, 210Pb and 40K from a coal ash and slag disposal site and control soil under field conditions: A preliminary study.

PubMed

Skoko, Božena; Marović, Gordana; Babić, Dinko; Šoštarić, Marko; Jukić, Mirela

2017-06-01

The aim of this study was to investigate the uptake of 238 U, 235 U, 232 Th, 226 Ra, 210 Pb and 40 K by plants that grow on a coal ash and slag disposal site known for its higher content of naturally occurring radionuclides. Plant species that were sampled are common for the Mediterranean flora and can be divided as follows: grasses & herbs, shrubs and trees. To compare the activity concentrations and the resultant concentration ratios of the disposal site with those in natural conditions, we used control data specific for the research area, obtained for plants growing on untreated natural soil. Radionuclide activity concentrations were determined by high resolution gamma-ray spectrometry. Media parameters (pH, electrical conductivity and organic matter content) were also analysed. We confirmed significantly higher activity concentrations of 238 U, 235 U, 226 Ra and 210 Pb in ash and slag compared to control soil. However, a significant increase in the radionuclide activity concentration in the disposal site's vegetation was observed only for 226 Ra. On the contrary, a significantly smaller activity concentration of 40 K in ash and slag had no impact on its activity concentration in plant samples. The calculated plant uptake of 238 U, 235 U, 226 Ra and 210 Pb is significantly smaller in comparison with the uptake at the control site, while it is vice versa for 40 K. No significant difference was observed between the disposal site and the control site's plant uptake of 232 Th. These results can be the foundation for further radioecological assessment of this disposal site but also, globally, they can contribute to a better understanding of nature and long-term management of such disposal sites. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biological responses to contaminants in darters (Etheostoma spp.) collected from rural and urban regions of the Grand River, ON, Canada.

PubMed

Diamond, Sam R; Sultana, Tamanna; Servos, Mark R; Metcalfe, Chris D

2016-09-01

Urban and agricultural activities may introduce chemical stressors, including contaminants of emerging concern (CECs) and current use pesticides (CUPs) into riverine systems. The objective of this study was to determine if fish collected from various sites in the Grand River, ON, Canada show biomarkers of exposure to these classes of contaminants, and if the biomarker patterns vary in fish collected from urbanized and agricultural sites. Female rainbow darters (Etheostoma caeruleum) and female fantail darters (Etheostoma flabellare) were collected from the Grand River in June, 2014 for biomarker analysis from two urbanized sites and three agricultural sites. Over the same period of time, Polar Organic Chemical Integrative Samplers (POCIS) were deployed for 2weeks at each site to monitor for the presence of CUPs and CECs. Data on the liver somatic index for darters indicate site-specific differences in this condition factor (p<0.05). Significant differences in the levels of thiobarbituric acid reactive substances (TBARS) in gill tissue (p<0.05) of darters collected from the various sites indicate site-specific differences in oxidative stress. The activities of ethoxyresorufin-O-deethylase (EROD) in the liver tissue of rainbow darters were significantly different between sites (p<0.05), indicating differences in exposure to chemicals that induce or inhibit CYP450 1A metabolic activity. Finally, acetylcholinesterase (AChE) activity in brain tissue was significantly different between rainbow darters collected from rural and urban sites (p<0.05). These data showing different impacts from chemical inputs related to land uses in the watershed may be useful in developing mitigation strategies to reduce impacts on fish and other aquatic organisms in receiving environments. Copyright © 2016. Published by Elsevier Inc.

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

The effects of intragrain defects on the local photoresponse of polycrystalline silicon solar cells

NASA Astrophysics Data System (ADS)

Inoue, N.; Wilmsen, C. W.; Jones, K. A.

1981-02-01

Intragrain defects in Wacker cast and Monsanto zone-refined polycrystalline silicon materials were investigated using the electron-beam-induced current (EBIC) technique. The EBIC response maps were compared with etch pit, local diffusion length and local photoresponse measurements. It was determined that the Wacker polycrystalline silicon has a much lower density of defects than does the Monsanto polycrystalline silicon and that most of the defects in the Wacker material are not active recombination sites. A correlation was found between the recombination site density, as determined by EBIC, and the local diffusion length. It is shown that a large density of intragrain recombination sites greatly reduces the minority carrier diffusion length and thus can significantly reduce the photoresponse of solar cells.

Receptor type I and type II binding regions and the peptidyl-prolyl isomerase site of cyclophilin B are required for enhancement of T-lymphocyte adhesion to fibronectin.

PubMed

Carpentier, Mathieu; Allain, Fabrice; Slomianny, Marie-Christine; Durieux, Sandrine; Vanpouille, Christophe; Haendler, Bernard; Spik, Geneviève

2002-04-23

Cyclophilin B (CyPB), a cyclosporin A (CsA) binding protein, interacts with two types of binding sites at the surface of T-lymphocytes. The type I sites correspond to functional receptors involved in endocytosis and the type II sites to sulfated glycosaminoglycans (GAGs). Mutational analysis of CyPB has revealed that W128, which is part of the CsA-binding pocket, is implicated in the binding to the functional type I receptors and that two amino acid clusters located in the N-terminus ensure the binding to GAGs. The peptidyl-prolyl isomerase activity of CyPB is not required for receptor binding. We have recently demonstrated that CyPB enhances adhesion of peripheral blood T-lymphocytes to fibronectin, a component of the extracellular matrix. We intended to identify additional amino acids involved in the binding of CyPB to its functional type I receptor and to determine regions responsible for the stimulation of peripheral blood T-lymphocyte adhesion. We determined that residues R76, G77, K132, D155, and D158 of the calcineurin (CN) interacting region were implicated in the recognition of type I receptor but not of GAGs. We also found that two different changes in the N-terminal extension that abated binding to GAGs prevented adhesion of peripheral blood T-lymphocytes to coated CyPB, whereas abbrogation of the PPIase activity had no effect. On the other hand, the adhesion of peripheral blood T-lymphocytes to coated fibronectin was not stimulated by CyPB mutants devoid of either type I receptor or GAGs binding activity or by mutants of the PPIase site. Altogether, the results demonstrate that different regions of CyPB are involved in peripheral blood T-lymphocyte activation and imply a novel important physiological function for peptidyl-prolyl isomerase activity.

Radionuclides (40K, 232Th and 238U) and Heavy Metals (Cr, Ni, Cu, Zn, As and Pb) Distribution Assessment at Renggam Landfill, Simpang Renggam, Johor, Malaysia

NASA Astrophysics Data System (ADS)

Zaidi, E.; FahrulRazi, MJ; Azhar, ATS; Hazreek, ZAM; Shakila, A.; Norshuhaila, MS; Omeje, M.

2017-08-01

The assessment of radioactivity levels and the distribution of heavy metals in soil samples at CEP Farm landfill, Renggam in Johor State was to determine the activity concentrations of naturally occurring radionuclides and heavy metal concentrations of this landfill. The background radiation was monitored to estimate the exposure level. The activity concentrations of radionuclides in soil samples were determined using HPGe gamma ray spectroscopy whereas the heavy metal concentration was measured using X-RF analysis. The mean exposure rate at the landfill site was 36.2±2.4 μR hr-1 and the annual effective dose rate at the landfill site was 3.19 ± 0.22 mSv yr-1. However, residential area has lower mean exposure dose rate of about 16.33±0.72 μR hr-1 and has an annual effective dose rate of 1.43±0.06 mSv yr-1 compared to landfill sites. The mean activity concentration of 40K, 238U and 232Th at landfill site were 239.95±15.89 Bq kg-1, 20.90±2.49 Bq kg-1 and 40.61±4.59 Bq kg-1, respectively. For heavy metal compositions, Cr, Ni and Cu have mean concentration of 232±10 ppm, 23±2 ppm, and 46±19 ppm, respectively. Whereas, Zn has concentration of 64±9 ppm and concentration of 12±1 ppm and 71±2 ppm was estimated for As and Pb respectively. The higher activity concentration of 40K down the slope through leaching process whereas the higher activity level of 238U content at the landfill site may be attributed to the soil disruption to local equilibrium.

Left Ventricular Lead Placement Targeted at the Latest Activated Site Guided by Electrophysiological Mapping in Coronary Sinus Branches Improves Response to Cardiac Resynchronization Therapy.

PubMed

Liang, Yanchun; Yu, Haibo; Zhou, Weiwei; Xu, Guoqing; Sun, Y I; Liu, Rong; Wang, Zulu; Han, Yaling

2015-12-01

Electrophysiological mapping (EPM) in coronary sinus (CS) branches is feasible for guiding LV lead placement to the optimal, latest activated site at cardiac resynchronization therapy (CRT) procedures. However, whether this procedure optimizes the response to CRT has not been demonstrated. This study was to evaluate effects of targeting LV lead at the latest activated site guided by EPM during CRT. Seventy-six consecutive patients with advanced heart failure who were referred for CRT were divided into mapping (MG) and control groups (CG). In MG, the LV lead, also used as a mapping bipolar electrode, was placed at the latest activated site determined by EPM in CS branches. In CG, conventional CRT procedure was performed. Patients were followed for 6 months after CRT. Baseline characteristics were comparable between the 2 groups. In MG (n = 29), EPM was successfully performed in 85 of 91 CS branches during CRT. A LV lead was successfully placed at the latest activated site guided by EPM in 27 (93.1%) patients. Compared with CG (n = 47), MG had a significantly higher rate (86.2% vs. 63.8%, P = 0.039) of response (>15% reduction in LV end-systolic volume) to CRT, a higher percentage of patients with clinical improvement of ≥2 NYHA functional classes (72.4% vs. 44.7%, P = 0.032), and a shorter QRS duration (P = 0.004). LV lead placed at the latest activated site guided by EPM resulted in a significantly greater CRT response, and a shorter QRS duration. © 2015 Wiley Periodicals, Inc.

Direct instrumental identification of catalytically active surface sites

NASA Astrophysics Data System (ADS)

Pfisterer, Jonas H. K.; Liang, Yunchang; Schneider, Oliver; Bandarenka, Aliaksandr S.

2017-09-01

The activity of heterogeneous catalysts—which are involved in some 80 per cent of processes in the chemical and energy industries—is determined by the electronic structure of specific surface sites that offer optimal binding of reaction intermediates. Directly identifying and monitoring these sites during a reaction should therefore provide insight that might aid the targeted development of heterogeneous catalysts and electrocatalysts (those that participate in electrochemical reactions) for practical applications. The invention of the scanning tunnelling microscope (STM) and the electrochemical STM promised to deliver such imaging capabilities, and both have indeed contributed greatly to our atomistic understanding of heterogeneous catalysis. But although the STM has been used to probe and initiate surface reactions, and has even enabled local measurements of reactivity in some systems, it is not generally thought to be suited to the direct identification of catalytically active surface sites under reaction conditions. Here we demonstrate, however, that common STMs can readily map the catalytic activity of surfaces with high spatial resolution: we show that by monitoring relative changes in the tunnelling current noise, active sites can be distinguished in an almost quantitative fashion according to their ability to catalyse the hydrogen-evolution reaction or the oxygen-reduction reaction. These data allow us to evaluate directly the importance and relative contribution to overall catalyst activity of different defects and sites at the boundaries between two materials. With its ability to deliver such information and its ready applicability to different systems, we anticipate that our method will aid the rational design of heterogeneous catalysts.

Role of Architecture in the Function and Specificity of Two Notch-Regulated Transcriptional Enhancer Modules

PubMed Central

Liu, Feng; Posakony, James W.

2012-01-01

In Drosophila melanogaster, cis-regulatory modules that are activated by the Notch cell–cell signaling pathway all contain two types of transcription factor binding sites: those for the pathway's transducing factor Suppressor of Hairless [Su(H)] and those for one or more tissue- or cell type–specific factors called “local activators.” The use of different “Su(H) plus local activator” motif combinations, or codes, is critical to ensure that only the correct subset of the broadly utilized Notch pathway's target genes are activated in each developmental context. However, much less is known about the role of enhancer “architecture”—the number, order, spacing, and orientation of its component transcription factor binding motifs—in determining the module's specificity. Here we investigate the relationship between architecture and function for two Notch-regulated enhancers with spatially distinct activities, each of which includes five high-affinity Su(H) sites. We find that the first, which is active specifically in the socket cells of external sensory organs, is largely resistant to perturbations of its architecture. By contrast, the second enhancer, active in the “non-SOP” cells of the proneural clusters from which neural precursors arise, is sensitive to even simple rearrangements of its transcription factor binding sites, responding with both loss of normal specificity and striking ectopic activity. Thus, diverse cryptic specificities can be inherent in an enhancer's particular combination of transcription factor binding motifs. We propose that for certain types of enhancer, architecture plays an essential role in determining specificity, not only by permitting factor–factor synergies necessary to generate the desired activity, but also by preventing other activator synergies that would otherwise lead to unwanted specificities. PMID:22792075

Oncoprotein protein kinase

DOEpatents

Karin, M.; Hibi, M.; Lin, A.

1997-02-25

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE is disclosed. The polypeptide has serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences. The method of detection of JNK is also provided. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites. 44 figs.

INVESTIGATIONS ON THE ANTIGENICITY OF SNAKE VENOMS

DTIC Science & Technology

The chemical composition of viperotoxin, the neurotoxic protein isolated from the venom of Vipera palestinae has been determined. Viperotoxin is...carboxy-terminal position of the viperotoxin chain. Vipera palestinae hemorrhagin has been purified and isolated. Further tests have established that...active sites, or selective blocking of a part of one active center having two distinct biological activities. Purified neurotoxin of V. palestinae

Oncoprotein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

2001-02-27

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Oncoprotein protein kinase

DOEpatents

Karin, Michael; Hibi, Masahiko; Lin, Anning

1999-01-01

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction.

PubMed

Saini, Vikas; Marchese, Adriano; Tang, Wei-Jen; Majetschak, Matthias

2011-12-23

Ubiquitin, a post-translational protein modifier inside the cell, functions as a CXC chemokine receptor (CXCR) 4 agonist outside the cell. However, the structural determinants of the interaction between extracellular ubiquitin and CXCR4 remain unknown. Utilizing C-terminal truncated ubiquitin and ubiquitin mutants, in which surface residues that are known to interact with ubiquitin binding domains in interacting proteins are mutated (Phe-4, Leu-8, Ile-44, Asp-58, Val-70), we provide evidence that the ubiquitin-CXCR4 interaction follows a two-site binding mechanism in which the hydrophobic surfaces surrounding Phe-4 and Val-70 are important for receptor binding, whereas the flexible C terminus facilitates receptor activation. Based on these findings and the available crystal structures, we then modeled the ubiquitin-CXCR4 interface with the RosettaDock software followed by small manual adjustments, which were guided by charge complementarity and anticipation of a conformational switch of CXCR4 upon activation. This model suggests three residues of CXCR4 (Phe-29, Phe-189, Lys-271) as potential interaction sites. Binding studies with HEK293 cells overexpressing wild type and CXCR4 after site-directed mutagenesis confirm that these residues are important for ubiquitin binding but that they do not contribute to the binding of stromal cell-derived factor 1α. Our findings suggest that the structural determinants of the CXCR4 agonist activity of ubiquitin mimic the typical structure-function relationship of chemokines. Furthermore, we provide evidence for separate and specific ligand binding sites on CXCR4. As exogenous ubiquitin has been shown to possess therapeutic potential, our findings are expected to facilitate the structure-based design of new compounds with ubiquitin-mimetic actions on CXCR4.

Comprehensive meta-analysis of Signal Transducers and Activators of Transcription (STAT) genomic binding patterns discerns cell-specific cis-regulatory modules

PubMed Central

2013-01-01

Background Cytokine-activated transcription factors from the STAT (Signal Transducers and Activators of Transcription) family control common and context-specific genetic programs. It is not clear to what extent cell-specific features determine the binding capacity of seven STAT members and to what degree they share genetic targets. Molecular insight into the biology of STATs was gained from a meta-analysis of 29 available ChIP-seq data sets covering genome-wide occupancy of STATs 1, 3, 4, 5A, 5B and 6 in several cell types. Results We determined that the genomic binding capacity of STATs is primarily defined by the cell type and to a lesser extent by individual family members. For example, the overlap of shared binding sites between STATs 3 and 5 in T cells is greater than that between STAT5 in T cells and non-T cells. Even for the top 1,000 highly enriched STAT binding sites, ~15% of STAT5 binding sites in mouse female liver are shared by other STATs in different cell types while in T cells ~90% of STAT5 binding sites are co-occupied by STAT3, STAT4 and STAT6. In addition, we identified 116 cis-regulatory modules (CRM), which are recognized by all STAT members across cell types defining a common JAK-STAT signature. Lastly, in liver STAT5 binding significantly coincides with binding of the cell-specific transcription factors HNF4A, FOXA1 and FOXA2 and is associated with cell-type specific gene transcription. Conclusions Our results suggest that genomic binding of STATs is primarily determined by the cell type and further specificity is achieved in part by juxtaposed binding of cell-specific transcription factors. PMID:23324445

Protein arginine deiminase 2 binds calcium in an ordered fashion: Implications for inhibitor design

DOE PAGES

Slade, Daniel J.; Fang, Pengfei; Dreyton, Christina J.; ...

2015-01-26

Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ionsmore » that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.« less

Protein Arginine Deiminase 2 Binds Calcium in an Ordered Fashion: Implications for Inhibitor Design

PubMed Central

2015-01-01

Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ions that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs. PMID:25621824

A macroinvertebrate assessment of Ozark streams located in lead-zinc mining areas of the Viburnum Trend in southeastern Missouri, USA

USGS Publications Warehouse

Poulton, Barry C.; Allert, Ann L.; Besser, John M.; Schmitt, Christopher J.; Brumbaugh, William G.; Fairchild, James F.

2010-01-01

The Viburnum Trend lead-zinc mining subdistrict is located in the southeast Missouri portion of the Ozark Plateau. In 2003 and 2004, we assessed the ecological effects of mining in several watersheds in the region. We included macroinvertebrate surveys, habitat assessments, and analysis of metals in sediment, pore water, and aquatic biota. Macroinvertebrates were sampled at 21 sites to determine aquatic life impairment status (full, partial, or nonsupport) and relative biotic condition scores. Macroinvertebrate biotic condition scores were significantly correlated with cadmium, nickel, lead, zinc, and specific conductance in 2003 (r = -0.61 to -0.68) and with cadmium, lead, and pore water toxic units in 2004 (r = -0.55 to -0.57). Reference sites were fully supporting of aquatic life and had the lowest metals concentrations and among the highest biotic condition scores in both years. Sites directly downstream from mining and related activities were partially supporting, with biotic condition scores 10% to 58% lower than reference sites. Sites located greater distances downstream from mining activities had intermediate scores and concentrations of metals. Results indicate that elevated concentrations of metals originating from mining activities were the underlying cause of aquatic life impairment in several of the streams studied. There was general concurrence among the adversely affected sites in how the various indicators responded to mining activities during the overall study.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diaz, A.; Valdez, V; Rudino-Pinera, E

Neurospora crassa has two large-subunit catalases, CAT-1 and CAT-3. CAT-1 is associated with non-growing cells and accumulates particularly in asexual spores; CAT-3 is associated with growing cells and is induced under different stress conditions. It is our interest to elucidate the structure-function relationships in large-subunit catalases. Here we have determined the CAT-3 crystal structure and compared it with the previously determined CAT-1 structure. Similar to CAT-1, CAT-3 hydrogen peroxide (H{sub 2}O{sub 2}) saturation kinetics exhibited two components, consistent with the existence of two active sites: one saturated in the millimolar range and the other in the molar range. In themore » CAT-1 structure, we found three interesting features related to its unusual kinetics: (a) a constriction in the channel that conveys H{sub 2}O{sub 2} to the active site; (b) a covalent bond between the tyrosine, which forms the fifth coordination bound to the iron of the heme, and a vicinal cysteine; (c) oxidation of the pyrrole ring III to form a cis-hydroxyl group in C5 and a cis-{gamma}-spirolactone in C6. The site of heme oxidation marks the starts of the central channel that communicates to the central cavity and the shortest way products can exit the active site. CAT-3 has a similar constriction in its major channel, which could function as a gating system regulated by the H{sub 2}O{sub 2} concentration before the gate. CAT-3 functional tyrosine is not covalently bonded, but has instead the electron relay mechanism described for the human catalase to divert electrons from it. Pyrrole ring III in CAT-3 is not oxidized as it is in other large-subunit catalases whose structure has been determined. Different in CAT-3 from these enzymes is an occupied central cavity. Results presented here indicate that CAT-3 and CAT-1 enzymes represent a functional group of catalases with distinctive structural characteristics that determine similar kinetics.« less

Use of octopus as a bioindicator species: Baseline studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holdway, D.A.; Butty, J.S.; Brennan, S.E.

1995-12-31

The Australian octopus Octopus pallidus, is abundant, territorial, has a large digestive gland. This study was undertaken to assess octopii as a potential bioindicator species by establishing the efficacy of capturing octopi using traplines, and determining the impact of various modifying factors on the activities of digestive gland mixed-function oxidase (MFO) enzymes including ethoxyresorufin O-deethylase (EROD), ethoxycoumarin O-deethylase (ECOD) and total P-450. Trap success rates in Port Phillip Bay were 15--28% for the ``potentially contaminated`` site and 85% for the reference site. Cytochrome P-450 showed significant seasonal differences, with no site or sex differences. Mean ({+-} SE) Autumn P-450 valuesmore » of 74.8 ({+-}5.5) pmol/mg protein were higher than Winter values of 51.2 ({+-}7.6), which were higher than Spring values of 21.8 ({+-}4.0) pmol/mg protein. Summer P-450 values of 61.4 ({+-} 9.8) pmol/mg protein were only different from Spring values. Mean ({+-} SE) Spring ECOD activity of 3.3 ({+-} 0.7) pmol/min/mg protein was lower than Summer, Autumn and Winter ECOD values of 8.9 ({+-} 1.6) 6.5 ({+-} 1.2) and 8.6 ({+-} 2.3) pmol/min/mg protein respectively. Females had roughly half the ECOD activities of males (3.8 {+-} 0.8 compared to 7.4 {+-} 0.9 pmol/min/mg protein). All octopi digestive gland EROD activities were low (roughly 0.2 pmol/min/mg protein) with no sex, site nor seasonal differences. Potential for using octopus as a bioindicator appears promising but sensitivity to chemical exposure has yet to be determined.« less

Mechanism of SO2 removal by carbon

USGS Publications Warehouse

Lizzio, Anthony A.; DeBarr, Joseph A.

1997-01-01

The reaction of SO2 with carbon (C) in the presence of O2 and H2O involves a series of reactions that leads to the formation of sulfuric acid as the final product. The rate-determining step in the overall process is the oxidation of SO2 to SO3. Three SO2 oxidation reactions are possible. Adsorbed SO2 (C−SO2) can react either with gas phase O2 or with adsorbed oxygen (C−O complex) to form sulfur trioxide (SO3), or gas phase SO2 can react directly with the C−O complex. In optimizing the SO2 removal capabilities of carbon, most studies only assume a given mechanism for SO2 adsorption and conversion to H2SO4 to be operable. The appropriate SO2 oxidation step and role of the C−O complex in this mechanism remain to be determined. The ultimate goal of this study was to prepare activated char from Illinois coal with optimal properties for low-temperature (80−150°C) removal of sulfur dioxide from coal combustion flue gas. The SO2 adsorption capacity of activated char was found to be inversely proportional to the amount of oxygen adsorbed on its surface. A temperature-programmed desorption technique was developed to titrate those sites responsible for adsorption of SO2 and conversion to H2SO4. On the basis of these results, a mechanism for SO2 removal by carbon was proposed. The derived rate expression showed SO2 adsorption to be dependent only on the fundamental rate constant and concentration of carbon atoms designated as free sites. Recent studies indicate a similar relationship exists between the rate of carbon gasification (in CO2 or H2O) and the number of reactive sites as determined by transient kinetics experiments. Utilizing the concept of active or free sites, it was possible to produce a char from Illinois coal having an SO2 adsorption capacity surpassing that of a commercial catalytic activated carbon.

The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II.

PubMed

Buermeyer, A B; Thompson, N E; Strasheim, L A; Burgess, R R; Farnham, P J

1992-05-01

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.

Verification survey report of the south waste tank farm training/test tower and hazardous waste storage lockers at the West Valley demonstration project, West Valley, New York

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weaver, Phyllis C.

A team from ORAU's Independent Environmental Assessment and Verification Program performed verification survey activities on the South Test Tower and four Hazardous Waste Storage Lockers. Scan data collected by ORAU determined that both the alpha and alpha-plus-beta activity was representative of radiological background conditions. The count rate distribution showed no outliers that would be indicative of alpha or alpha-plus-beta count rates in excess of background. It is the opinion of ORAU that independent verification data collected support the site?s conclusions that the South Tower and Lockers sufficiently meet the site criteria for release to recycle and reuse.

Site characterization progress report: Yucca Mountain, Nevada, October 1, 1994--March 31, 1995, Number 12. Nuclear Waste Policy Act (Section 113)

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1995-08-01

During the first half of fiscal year 1995, most activities at the Yucca Mountain Site Characterization Project were directed at implementing the Program Plan developed by the Office of Civilian Radioactive Waste Management. The Plan is designed to enable the Office to make measurable and significant progress toward key objectives over the next five years within the financial resources that can be realistically expected. Activities this period focused on the immediate goal of determining by 1998 whether Yucca Mountain, Nevada, is technically suitable as a possible site for a geologic repository for the permanent disposal of spent nuclear fuel andmore » high-level radioactive waste. Work on the Project advanced in several critical areas, including programmatic activities such as issuing the Program Plan, completing the first technical basis report to support the assessment of three 10 CFR 960 guidelines, developing the Notice of Intent for the Environmental Impact Statement, submitting the License Application Annotated Outline, and beginning a rebaselining effort to conform with the goals of the Program Plan. Scientific investigation and analysis of the site and design and construction activities to support the evaluation of the technical suitability of the site also advanced. Specific details relating to all Project activities and reports generated are presented in this report.« less

Computational active site analysis of molecular pathways to improve functional classification of enzymes.

PubMed

Ozyurt, A Sinem; Selby, Thomas L

2008-07-01

This study describes a method to computationally assess the function of homologous enzymes through small molecule binding interaction energy. Three experimentally determined X-ray structures and four enzyme models from ornithine cyclo-deaminase, alanine dehydrogenase, and mu-crystallin were used in combination with nine small molecules to derive a function score (FS) for each enzyme-model combination. While energy values varied for a single molecule-enzyme combination due to differences in the active sites, we observe that the binding energies for the entire pathway were proportional for each set of small molecules investigated. This proportionality of energies for a reaction pathway appears to be dependent on the amino acids in the active site and their direct interactions with the small molecules, which allows a function score (FS) to be calculated to assess the specificity of each enzyme. Potential of mean force (PMF) calculations were used to obtain the energies, and the resulting FS values demonstrate that a measurement of function may be obtained using differences between these PMF values. Additionally, limitations of this method are discussed based on: (a) larger substrates with significant conformational flexibility; (b) low homology enzymes; and (c) open active sites. This method should be useful in accurately predicting specificity for single enzymes that have multiple steps in their reactions and in high throughput computational methods to accurately annotate uncharacterized proteins based on active site interaction analysis. 2008 Wiley-Liss, Inc.

Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis.

PubMed

Bisht, Shveta; Rajaram, Venkatesan; Bharath, Sakshibeedu R; Kalyani, Josyula Nitya; Khan, Farida; Rao, Appaji N; Savithri, Handanahal S; Murthy, Mathur R N

2012-06-08

Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp(120) and Lys(77) is suggested.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.

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

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

NASA Astrophysics Data System (ADS)

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

2015-05-01

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

Crystal Structure of the Dithiol Oxidase DsbA Enzyme from Proteus Mirabilis Bound Non-covalently to an Active Site Peptide Ligand

PubMed Central

Kurth, Fabian; Duprez, Wilko; Premkumar, Lakshmanane; Schembri, Mark A.; Fairlie, David P.; Martin, Jennifer L.

2014-01-01

The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery. PMID:24831013

Crystallographic structure of a small molecule SIRT1 activator-enzyme complex

NASA Astrophysics Data System (ADS)

Dai, Han; Case, April W.; Riera, Thomas V.; Considine, Thomas; Lee, Jessica E.; Hamuro, Yoshitomo; Zhao, Huizhen; Jiang, Yong; Sweitzer, Sharon M.; Pietrak, Beth; Schwartz, Benjamin; Blum, Charles A.; Disch, Jeremy S.; Caldwell, Richard; Szczepankiewicz, Bruce; Oalmann, Christopher; Yee Ng, Pui; White, Brian H.; Casaubon, Rebecca; Narayan, Radha; Koppetsch, Karsten; Bourbonais, Francis; Wu, Bo; Wang, Junfeng; Qian, Dongming; Jiang, Fan; Mao, Cheney; Wang, Minghui; Hu, Erding; Wu, Joe C.; Perni, Robert B.; Vlasuk, George P.; Ellis, James L.

2015-07-01

SIRT1, the founding member of the mammalian family of seven NAD+-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key intermolecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases.

Retrotransposon Tf1 is targeted to pol II promoters by transcription activators

PubMed Central

Leem, Young-Eun; Ripmaster, Tracy; Kelly, Felice; Ebina, Hirotaka; Heincelman, Marc; Zhang, Ke; Grewal, Shiv I. S.; Hoffman, Charles S.; Levin, Henry L.

2008-01-01

SUMMARY The LTR-retrotransposon Tf1 preserves the coding capacity of its host Schizosaccharomyces pombe by integrating upstream of open reading frames (ORFs). To determine which features of the target sites were recognized by the transposon, we introduced plasmids containing candidate insertion sites into S. pombe and mapped the positions of integration. We found that Tf1 was targeted specifically to the promoters of pol II transcribed genes. A detailed analysis of integration in plasmids that contained either ade6 or fbp1 revealed insertions occurred in the promoters at positions where transcription factors bound. Further experiments revealed that the activator Atf1p and its binding site were required for directing integration to the promoter of fbp1. An interaction between Tf1 integrase and Atf1p was observed indicating that integration at fbp1 was mediated by the activator bound to its promoter. Surprisingly we found Tf1 contained sequences that activated transcription and these substituted for elements of the ade6 promoter disrupted by integration. PMID:18406330

Retrotransposon Tf1 is targeted to Pol II promoters by transcription activators.

PubMed

Leem, Young-Eun; Ripmaster, Tracy L; Kelly, Felice D; Ebina, Hirotaka; Heincelman, Marc E; Zhang, Ke; Grewal, Shiv I S; Hoffman, Charles S; Levin, Henry L

2008-04-11

The LTR-retrotransposon Tf1 preserves the coding capacity of its host Schizosaccharomyces pombe by integrating upstream of open reading frames (ORFs). To determine which features of the target sites were recognized by the transposon, we introduced plasmids containing candidate insertion sites into S. pombe and mapped the positions of integration. We found that Tf1 was targeted specifically to the promoters of Pol II-transcribed genes. A detailed analysis of integration in plasmids that contained either ade6 or fbp1 revealed insertions occurred in the promoters at positions where transcription factors bound. Further experiments revealed that the activator Atf1p and its binding site were required for directing integration to the promoter of fbp1. An interaction between Tf1 integrase and Atf1p was observed, indicating that integration at fbp1 was mediated by the activator bound to its promoter. Surprisingly, we found Tf1 contained sequences that activated transcription, and these substituted for elements of the ade6 promoter disrupted by integration.

Kinetic analysis of butyrylcholinesterase-catalyzed hydrolysis of acetanilides.

PubMed

Masson, Patrick; Froment, Marie-Thérèse; Gillon, Emilie; Nachon, Florian; Darvesh, Sultan; Schopfer, Lawrence M

2007-09-01

The aryl-acylamidase (AAA) activity of butyrylcholinesterase (BuChE) has been known for a long time. However, the kinetic mechanism of aryl-acylamide hydrolysis by BuChE has not been investigated. Therefore, the catalytic properties of human BuChE and its peripheral site mutant (D70G) toward neutral and charged aryl-acylamides were determined. Three neutral (o-nitroacetanilide, m-nitroacetanilide, o-nitrophenyltrifluoroacetamide) and one positively charged (3-(acetamido) N,N,N-trimethylanilinium, ATMA) acetanilides were studied. Hydrolysis of ATMA by wild-type and D70G enzymes showed a long transient phase preceding the steady state. The induction phase was characterized by a hysteretic "burst". This reflects the existence of two enzyme states in slow equilibrium with different catalytic properties. Steady-state parameters for hydrolysis of the three acetanilides were compared to catalytic parameters for hydrolysis of esters giving the same acetyl intermediate. Wild-type BuChE showed substrate activation while D70G displayed a Michaelian behavior with ATMA as with positively charged esters. Owing to the low affinity of BuChE for amide substrates, the hydrolysis kinetics of neutral amides was first order. Acylation was the rate-determining step for hydrolysis of aryl-acetylamide substrates. Slow acylation of the enzyme, relative to that by esters may, in part, be due suboptimal fit of the aryl-acylamides in the active center of BuChE. The hypothesis that AAA and esterase active sites of BuChE are non-identical was tested with mutant BuChE. It was found that mutations on the catalytic serine, S198C and S198D, led to complete loss of both activities. The silent variant (FS117) had neither esterase nor AAA activity. Mutation in the peripheral site (D70G) had the same effect on esterase and AAA activities. Echothiophate inhibited both activities identically. It was concluded that the active sites for esterase and AAA activities are identical, i.e. S198. This excludes any other residue present in the gorge for being the catalytic nucleophile pole.

Deconstructing thermodynamic parameters of a coupled system from site-specific observables.

PubMed

Chowdhury, Sandipan; Chanda, Baron

2010-11-02

Cooperative interactions mediate information transfer between structural domains of a protein molecule and are major determinants of protein function and modulation. The prevalent theories to understand the thermodynamic origins of cooperativity have been developed to reproduce the complex behavior of a global thermodynamic observable such as ligand binding or enzyme activity. However, in most cases the measurement of a single global observable cannot uniquely define all the terms that fully describe the energetics of the system. Here we establish a theoretical groundwork for analyzing protein thermodynamics using site-specific information. Our treatment involves extracting a site-specific parameter (defined as χ value) associated with a structural unit. We demonstrate that, under limiting conditions, the χ value is related to the direct interaction terms associated with the structural unit under observation and its intrinsic activation energy. We also introduce a site-specific interaction energy term (χ(diff)) that is a function of the direct interaction energy of that site with every other site in the system. When combined with site-directed mutagenesis and other molecular level perturbations, analyses of χ values of site-specific observables may provide valuable insights into protein thermodynamics and structure.

Determination of the Bridging Ligand in the Active Site of Tyrosinase.

PubMed

Zou, Congming; Huang, Wei; Zhao, Gaokun; Wan, Xiao; Hu, Xiaodong; Jin, Yan; Li, Junying; Liu, Junjun

2017-10-28

Tyrosinase is a type-3 copper enzyme that is widely distributed in plants, fungi, insects, and mammals. Developing high potent inhibitors against tyrosinase is of great interest in diverse fields including tobacco curing, food processing, bio-insecticides development, cosmetic development, and human healthcare-related research. In the crystal structure of Agaricus bisporus mushroom tyrosinase, there is an oxygen atom bridging the two copper ions in the active site. It is unclear whether the identity of this bridging oxygen is a water molecule or a hydroxide anion. In the present study, we theoretically determine the identity of this critical bridging oxygen by performing first-principles hybrid quantum mechanics/molecular mechanics/Poisson-Boltzmann-surface area (QM/MM-PBSA) calculations along with a thermodynamic cycle that aim to improve the accuracy. Our results show that the binding with water molecule is energy favored and the QM/MM-optimized structure is very close to the crystal structure, whereas the binding with hydroxide anions causes the increase of energy and significant structural changes of the active site, indicating that the identity of the bridging oxygen must be a water molecule rather than a hydroxide anion. The different binding behavior between water and hydroxide anions may explain why molecules with a carboxyl group or too many negative charges have lower inhibitory activity. In light of this, the design of high potent active inhibitors against tyrosinase should satisfy both the affinity to the copper ions and the charge neutrality of the entire molecule.

A Quasi-Laue Neutron Crystallographic Study of D-Xylose Isomerase

NASA Technical Reports Server (NTRS)

Meilleur, Flora; Snell, Edward H.; vanderWoerd, Mark; Judge, Russell A.; Myles, Dean A. A.

2006-01-01

Hydrogen atom location and hydrogen bonding interaction determination are often critical to explain enzymatic mechanism. Whilst it is difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (less than 1.0 Angstrom) resolution data available, neutron crystallography provides an experimental tool to directly localise hydrogeddeuteriwn atoms in biological macromolecules at resolution of 1.5-2.0 Angstroms. Linearisation and isomerisation of xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data were collected on Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism (Myles et al. 1998). The neutron structure unambiguously reveals the protonation state of His 53 in the active site, identifying the model for the enzymatic pathway.

The crystal structure of Erwinia amylovora levansucrase provides a snapshot of the products of sucrose hydrolysis trapped into the active site.

PubMed

Wuerges, Jochen; Caputi, Lorenzo; Cianci, Michele; Boivin, Stephane; Meijers, Rob; Benini, Stefano

2015-09-01

Levansucrases are members of the glycoside hydrolase family and catalyse both the hydrolysis of the substrate sucrose and the transfer of fructosyl units to acceptor molecules. In the presence of sufficient sucrose, this may either lead to the production of fructooligosaccharides or fructose polymers. Aim of this study is to rationalise the differences in the polymerisation properties of bacterial levansucrases and in particular to identify structural features that determine different product spectrum in the levansucrase of the Gram-negative bacterium Erwinia amylovora (Ea Lsc, EC 2.4.1.10) as compared to Gram-positive bacteria such as Bacillus subtilis levansucrase. Ea is an enterobacterial pathogen responsible for the Fire Blight disease in rosaceous plants (e.g., apple and pear) with considerable interest for the agricultural industry. The crystal structure of Ea Lsc was solved at 2.77 Å resolution and compared to those of other fructosyltransferases from Gram-positive and Gram-negative bacteria. We propose the structural features, determining the different reaction products, to reside in just a few loops at the rim of the active site funnel. Moreover we propose that loop 8 may have a role in product length determination in Gluconacetobacter diazotrophicus LsdA and Microbacterium saccharophilum FFase. The Ea Lsc structure shows for the first time the products of sucrose hydrolysis still bound in the active site. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

Multi-Modal Active Perception for Autonomously Selecting Landing Sites on Icy Moons

NASA Technical Reports Server (NTRS)

Arora, A.; Furlong, P. M.; Wong, U.; Fong, T.; Sukkarieh, S.

2017-01-01

Selecting suitable landing sites is fundamental to achieving many mission objectives in planetary robotic lander missions. However, due to sensing limitations, landing sites which are both safe and scientifically valuable often cannot be determined reliably from orbit, particularly, in icy moon missions where orbital sensing data is noisy and incomplete. This paper presents an active perception approach to Entry Descent and Landing (EDL) which enables the lander to autonomously plan informative descent trajectories, acquire high quality sensing data during descent and exploit this additional information to select higher utility landing sites. Our approach consists of two components: probabilistic modeling of landing site features and approximate trajectory planning using a sampling based planner. The proposed framework allows the lander to plan long horizons paths and remain robust to noisy data. Results in simulated environments show large performance improvements over alternative approaches and show promise that our approach has strong potential to improve science return of not only icy moon missions but EDL systems in general.

Prospective study of iliac crest bone graft harvest site pain and morbidity.

PubMed

Kim, David H; Rhim, Richard; Li, Ling; Martha, Juli; Swaim, Bryan H; Banco, Robert J; Jenis, Louis G; Tromanhauser, Scott G

2009-11-01

Morbidity associated with autologous bone graft harvest is an important factor in determining the utility of expensive alternatives such as recombinant bone morphogenic protein. The most frequently reported complication associated with graft harvest is chronic pain. To prospectively determine the degree of pain and morbidity associated with autologous iliac crest bone graft harvest and its effect on activities of daily living. Prospective observational cohort study. One hundred ten adult patients undergoing elective posterior lumbar spinal fusion surgery involving autologous iliac crest bone graft harvest. Patient self-reported Visual Analog Scale (VAS) scores for pain and a study-specific questionnaire regarding activities of daily living. One hundred ten patients were prospectively enrolled. Postoperative VAS scores (0-100) for harvest site pain were obtained at 6-week, 6- and 12-month follow-up. Patients completed a 12-month questionnaire regarding the persistence of specific symptoms and resulting limitation of specific activities. One hundred four patients were available for 1-year follow-up. Mean VAS pain scores (scale 0-100) at 6 weeks, 6 and 12 months were 22.7 (standard deviation [SD], 25.9), 15.9 (SD, 21.5), and 16.1 (SD, 24.6), respectively. At 12 months, 16.5% reported more severe pain from the harvest site than the primary surgical site, 29.1% reported numbness, and 11.3% found the degree of numbness bothersome, whereas 3.9% were bothered by scar appearance. With respect to activity limitations resulting from harvest site pain at 1 year, 15.1% reported some difficulty walking, 5.2% with employment, 12.9% with recreation, 14.1% with household chores, 7.6% with sexual activity, and 5.9% irritation from clothing. There is a significant rate of persistent pain and morbidity from iliac crest bone graft harvest when associated with elective spine surgery. Mean pain scores progressively decline over the first postoperative year. Nevertheless, harvest site pain remains functionally limiting in a significant percentage of patients 1 year after surgery. Rates of functional limitation are higher than previously reported and may be because of increased sensitivity of the prospective study design and targeted investigation of these specific symptoms. Validity of these findings is necessarily limited by patient ability to discriminate harvest site pain from alternative sources of back and buttock pain.

Ethenoguanines Undergo Glycosylation by Nucleoside 2′-Deoxyribosyltransferases at Non-Natural Sites

PubMed Central

Ye, Wenjie; Paul, Debamita; Gao, Lina; Seckute, Jolita; Jayaraj, Karupiah; Zhang, Zhenfa; Kaminski, P. Alexandre

2014-01-01

Deoxyribosyl transferases and functionally related purine nucleoside phosphorylases are used extensively for synthesis of non-natural deoxynucleosides as pharmaceuticals or standards for characterizing and quantitating DNA adducts. Hence exploring the conformational tolerance of the active sites of these enzymes is of considerable practical interest. We have determined the crystal structure at 2.1 Å resolution of Lactobacillus helveticus purine deoxyribosyl transferase (PDT) with the tricyclic purine 8,9-dihydro-9-oxoimidazo[2,1-b]purine (N 2,3-ethenoguanine) at the active site. The active site electron density map was compatible with four orientations, two consistent with sites for deoxyribosylation and two appearing to be unproductive. In accord with the crystal structure, Lactobacillus helveticus PDT glycosylates the 8,9-dihydro-9-oxoimidazo[2,1-b]purine at N7 and N1, with a marked preference for N7. The activity of Lactobacillus helveticus PDT was compared with that of the nucleoside 2′-deoxyribosyltransferase enzymes (DRT Type II) from Lactobacillus leichmannii and Lactobacillus fermentum, which were somewhat more effective in the deoxyribosylation than Lactobacillus helveticus PDT, glycosylating the substrate with product profiles dependent on the pH of the incubation. The purine nucleoside phosphorylase of Escherichia coli, also commonly used in ribosylation of non-natural bases, was an order of magnitude less efficient than the transferase enzymes. Modeling based on published active-site structures as templates suggests that in all cases, an active site Phe is critical in orienting the molecular plane of the purine derivative. Adventitious hydrogen bonding with additional active site residues appears to result in presentation of multiple nucleophilic sites on the periphery of the acceptor base for ribosylation to give a distribution of nucleosides. Chemical glycosylation of O 9-benzylated 8,9-dihydro-9-oxoimidazo[2,1-b]purine also resulted in N7 and N1 ribosylation. Absent from the enzymatic and chemical glycosylations is the natural pattern of N3 ribosylation, verified by comparison of spectroscopic and chromatographic properties with an authentic standard synthesized by an unambiguous route. PMID:25521390

Three-dimensional atomic arrangement around active/inactive dopant sites in boron-doped diamond

NASA Astrophysics Data System (ADS)

Kato, Yukako; Tsujikawa, Daichi; Hashimoto, Yusuke; Yoshida, Taisuke; Fukami, Shun; Matsuda, Hiroyuki; Taguchi, Munetaka; Matsushita, Tomohiro; Daimon, Hiroshi

2018-06-01

Boron-doped diamond has received significant attention as an advanced material for power devices owing to its high breakdown characteristics. To control the characteristics of diamond related to band conduction, it is essential to determine the atomic structure around dopants and to develop a method of controlling the atomic arrangement around dopants. We measured the photoelectron diffraction of a boron-doped diamond using a display-type ellipsoidal mesh analyzer to examine the dopant sites in heavily boron-doped diamond. The B 1s photoelectron spectrum shows two peaks for different chemical bonding sites. These two dopant sites were identified as the substitutional and interstitial sites in diamond.

Records of wells and chemical analyses of water from wells for the period June 13, 1984 to December 4, 1986 at the Maxey Flats Radioactive Waste Disposal Site, Kentucky

USGS Publications Warehouse

Lyverse, M.A.

1987-01-01

Lithologic data are presented for 113 wells drilled at the Maxey Flats Radioactive Waste Disposal Site for the period June 13, 1984 to December 4, 1986. Water levels, tritium concentrations, and specific conductance are also presented for wells yielding sufficient water for measuring and sampling. At least one sample was collected from most wells for the determination of gross alpha and beta activity. These activities and the results for gamma emitting radionuclides (Cobalt 60 and Cesium 137) are also presented. (USGS)

Functional microbial community response to nutrient pulses by artificial groundwater recharge practice in surface soils and subsoils.

PubMed

Schütz, Kirsten; Kandeler, Ellen; Nagel, Peter; Scheu, Stefan; Ruess, Liliane

2010-06-01

Subsurface microorganisms are essential constituents of the soil purification processes associated with groundwater quality. In particular, soil enzyme activity determines the biodegradation of organic compounds passing through the soil profile. Transects from surface soil to a depth of 3.5 m were investigated for microbial and chemical soil characteristics at two groundwater recharge sites and one control site. The functional diversity of the microbial community was analyzed via the activity of eight enzymes. Acid phosphomonoesterase was dominant across sites and depths, followed by L-leucine aminopeptidase and beta-glucosidase. Structural [e.g. phospholipid fatty acid (PLFA) pattern] and functional microbial diversities were linked to each other at the nonwatered site, whereas amendment with nutrients (DOC, NO(3)(-)) by flooding uncoupled this relationship. Microbial biomass did not differ between sites, whereas microbial respiration was the highest at the watered sites. Hence, excess nutrients available due to artificial groundwater recharge could not compensate for the limitation by others (e.g. phosphorus as assigned by acid phosphomonoesterase activity). Instead, at a similar microbial biomass, waste respiration via overflow metabolism occurred. In summary, ample supply of carbon by flooding led to a separation of decomposition and microbial growth, which may play an important role in regulating purification processes during groundwater recharge.

Epitope mapping of the domains of human angiotensin converting enzyme.

PubMed

Kugaevskaya, Elena V; Kolesanova, Ekaterina F; Kozin, Sergey A; Veselovsky, Alexander V; Dedinsky, Ilya R; Elisseeva, Yulia E

2006-06-01

Somatic angiotensin converting enzyme (sACE), contains in its single chain two homologous domains (called N- and C-domains), each bearing a functional zinc-dependent active site. The present study aims to define the differences between two sACE domains and to localize experimentally revealed antigenic determinants (B-epitopes) in the recently determined three-dimensional structure of testicular tACE. The predicted linear antigenic determinants of human sACE were determined by peptide scanning ("PEPSCAN") approach. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. Comparison of arrangement of epitopes in the human domains with the corresponding sequences of some mammalian sACEs enabled to classify the revealed antigenic determinants as variable or conserved areas. The location of antigenic determinants with respect to various structural elements and to functionally important sites of the human sACE C-domain was estimated. The majority of antigenic sites of the C-domain were located at the irregular elements and at the boundaries of secondary structure elements. The data show structural differences between the sACE domains. The experimentally revealed antigenic determinants were in agreement with the recently determined crystal tACE structure. New potential applications are open to successfully produce mono-specific and group-specific antipeptide antibodies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hyun You; Liu, Ping

Mixed metal oxides have attracted considerable attention in heterogeneous catalysis due to the unique stability, reactivity, and selectivity. Here, the activity and stability of the CuTiO x monolayer film supported on Cu(111), CuTiO x/Cu(111), during CO oxidation was explored using density functional theory (DFT). The unique structural frame of CuTiO x is able to stabilize and isolate a single Cu + site on the terrace, which is previously proposed active for CO oxidation. Furthermore, it is not the case, where the reaction via both the Langmuir–Hinshelwood (LH) and the Mars-van Krevelen (M-vK) mechanisms are hindered on such single Cu +more » site. Upon the formation of step-edges, the synergy among Cu δ+ sites, TiO x matrix, and Cu(111) is able to catalyze the reaction well. Depending on temperatures and partial pressure of CO and O 2, the surface structure varies, which determines the dominant mechanism. In accordance with our results, the Cu δ+ ion alone does not work well for CO oxidation in the form of single sites, while the synergy among multiple active sites is necessary to facilitate the reaction.« less

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.

Evaluating three trace metal contaminated sites: a field and laboratory investigation.

PubMed

Murray, P; Ge, Y; Hendershot, W H

2000-01-01

Selecting guidelines to evaluate elevated metals in urban brownfields is hindered by the lack of information for these sites on ecosystem structure and function. A study was performed to compare three trace metal-contaminated sites in the metropolitan Montreal area. The goal was to obtain an idea of the organisms that may be present on urban brownfields and to measure if elevated metals alter the presence and activity of the indigenous biota. Field and laboratory studies were conducted using simple methodologies to determine the extent to which microbial activity affected by trace metal content, to assess diversity of plant and soil invertebrate communities and to measure phytoaccumulation of trace metals. It was found that microbial activity, as measured by substrate-induced respiration (SIR) and nitrification, was not affected by the levels of soil Cd, Cu, Ni, Pb and Zn recorded on the sites. Seven of the 12 invertebrate groups collected were sampled on soils with similar Cd, Cu, Ni, Pb and Zn concentrations. Diversity of plant species increased as a function of the length of time the sites had been inactive. Levels of metals in plant tissue were influenced by soil characteristics and not by total soil Cd, Cu, Ni, Pb and Zn.

Complex catalytic behaviors of CuTiO x mixed-oxide during CO oxidation

DOE PAGES

Kim, Hyun You; Liu, Ping

2015-09-21

Mixed metal oxides have attracted considerable attention in heterogeneous catalysis due to the unique stability, reactivity, and selectivity. Here, the activity and stability of the CuTiO x monolayer film supported on Cu(111), CuTiO x/Cu(111), during CO oxidation was explored using density functional theory (DFT). The unique structural frame of CuTiO x is able to stabilize and isolate a single Cu + site on the terrace, which is previously proposed active for CO oxidation. Furthermore, it is not the case, where the reaction via both the Langmuir–Hinshelwood (LH) and the Mars-van Krevelen (M-vK) mechanisms are hindered on such single Cu +more » site. Upon the formation of step-edges, the synergy among Cu δ+ sites, TiO x matrix, and Cu(111) is able to catalyze the reaction well. Depending on temperatures and partial pressure of CO and O 2, the surface structure varies, which determines the dominant mechanism. In accordance with our results, the Cu δ+ ion alone does not work well for CO oxidation in the form of single sites, while the synergy among multiple active sites is necessary to facilitate the reaction.« less

Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morrison, W.J.

1988-01-01

The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF ormore » thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).« less

Tritium and radioactive carbon (14C) analyses of gas collected from unsaturated sediments next to a low-level radioactive-waste burial site south of Beatty, Nevada, April 1994 and July 1995

USGS Publications Warehouse

Prudic, David E.; Striegl, Robert G.

1995-01-01

Tritium activities in water vapor and radioactive carbon (14C) activities in carbon dioxide were determined in gas samples pumped from small-diameter air ports installed in a test hole within the unsaturated sediments next to a commercial burial site for low-level radioactive waste south of Beatty, Nevada. In April 1994, gas samples were collected from test hole UZB-2, which was drilled about 350 feet south of the southwest corner of the fence enclosing the burial site. The test hole is part of a study to determine the depth to which atmospheric air circulates through the unsaturated sediments at the desert site. Laboratory results completed in May 1995 show activities of tritium and 14C were greater than expected, with measured tritium in the water vapor as high as 762 tritium units at a depth of 79 feet and measured 14C in carbon dioxide as high as 1,700 percent modern carbon at a depth of 18 feet.In July 1995, the uppermost five air ports in test hole UZB-2 were resampled. In addition, water vapor was collected for tritium analyses at a distant test hole, and water vapor for tritium analyses and carbon dioxide for 14C analyses were collected from three depths at the research shaft about 200 feet north of test hole UZB-2, and at two shallow probes (depth of 5.5 feet) next to the fence enclosing the burial site. Analyses of samples collected in the upper 112 feet from test hole UZB-2 in July 1995 show the same distribution of tritium and 14C as analyses of samples collected in April 1994, except that activities were somewhat greater in July. The greatest activities of tritium and 14C were measured from a shallow probe next to the fence with activities of 29,400 tritium units and 517,000 percent modern carbon, respectively.

Resonance energy transfer between the active sites of creatine kinase from rabbit brain.

PubMed

Grossman, S H

1990-09-03

Resonance energy transfer was measured between the active site domains of the brain isozyme of creatine kinase (CK-BB). The reactive thiol near the active sites, one on each subunit of the dimeric protein, was derivatized using 5-[2-[iodoacetyl)amino)ethyl]aminonaphthalene-1-sulfonic acid (AED), 2-[4'-iodoacetamidoanilino]naphthalene-6-sulfonic acid (AANS) and 5-iodoacetamidofluorescein (AF). Suitable donor/acceptor protein conjugated hybrids were prepared by controlled kinetics producing CK-BB-AED/AF and CK-BB-AANS/AF. Transfer efficiencies, measured from the quenching of the donor lifetime and steady-state sensitized acceptor emission, ranged from 0.10 to 0.17. From determination of the donor/acceptor overlap integrals, donor quantum yields and attempts to delimit the orientation factor using steady-state and phase-resolved anisotropy measurements, it was found that a suitable estimate of the range between the active sites was between 45 and 57 A. This range is similar to that reported previously for the muscle isozyme of creatine kinase (Grossman, S.H. (1989) Biochemistry 28, 4894-4902) but is a significantly greater distance than detected for the hybrid, myocardial specific isozyme (Grossman, S.H. (1983) Biochemistry 22, 5369-5375).

Stereochemical study of mouse muscone receptor MOR215-1 and vibrational theory based on statistical physics formalism.

PubMed

Ben Khemis, Ismahene; Mechi, Nesrine; Ben Lamine, Abdelmottaleb

2018-02-10

In the biosensor system, olfactory receptor sites could be activated by odorant molecules and then the biological interactions are converted into electrical signals by a signal transduction cascade that leads the toopening of ion channels, generating a current that leads into the cilia and depolarizes the membrane. The aim of this paper is to present a new investigation that allows determining the olfactory band using a monolayer adsorption with identical sites modeling which may also describe the static and the dynamic sensitivities through the expression of the olfactory response. Moreover, knowing the size of receptor site in olfactory sensory neurons provides valuable information about the relationship between molecular structure and biological activity. The determination of microreceptors and mesoreceptors is mostly carried out via physical adsorption and the radius is calculated using the Kelvin equation. The mean values of radius obtained from the maximum of the receptor size distributions peaks are 4 nm for ℓ-muscone and 6 nm for d-muscone. Copyright © 2018. Published by Elsevier Ltd.

In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites

PubMed Central

Grey, Corinne; Clément, Julie A.J.; Buard, Jérôme; Leblanc, Benjamin; Gut, Ivo; Gut, Marta; Duret, Laurent

2017-01-01

In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed an extensive analysis of PRDM9 binding in mouse spermatocytes. Unexpectedly, we identified a noncanonical recruitment of PRDM9 to sites that lack recombination activity and the PRDM9 binding consensus motif. These sites include gene promoters, where PRDM9 is recruited in a DSB-dependent manner. Another subset reveals DSB-independent interactions between PRDM9 and genomic sites, such as the binding sites for the insulator protein CTCF. We propose that these DSB-independent sites result from interactions between hotspot-bound PRDM9 and genomic sequences located on the chromosome axis. PMID:28336543

EROD activity measured in flatfish from the area of the Sea Empress oil spill.

PubMed

Kirby, M F; Neall, P; Tylor, T

1999-05-01

Dab (Limanda limanda) and plaice (Pleuronectes platessa) were collected at five stations near to the site of the Sea Empress oil spill within two weeks of the incident and a further fourteen stations three months after the spillage. Ethoxyresorufin-O-deethylase (EROD) activity was determined in the livers of the specimens to determine whether induction could be detected. Statistically significant inter-site differences in EROD levels in both species were demonstrated. Elevated levels of EROD activity in dab were found at the two stations nearest to the incident up to three months after the spill but no clear relationship to putative contaminant levels was determined. EROD levels in plaice showed a generally similar pattern of induction as in dab. Correlation of EROD levels with other variables showed that sexual maturity had the greatest influence on dab during the study period. The plaice specimens were sexually immature and, therefore, did not demonstrate a corresponding relationship. It was concluded that, for EROD monitoring purposes, fish should be sampled during their sexually inactive phase and that close attention needs to be paid to other variables (depth, temperature, GSI, length, influential contaminants etc.) when interpreting the results.

Structural Model for the Flip-Flop Action in Thiamin Pyrophosphate-Dependent Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Dominiak, Paulina

2003-01-01

The derivative of vitamin B1 thiamin pyrophosphate (TPP) is a cofactor of enzymes performing catalysis in pathways of energy production, including (i) decarboxylation of alpha-keto acids followed by (ii) transketolation. These enzymes have shown a common mechanism of TPP activation by imposing an active V-conformation of this coenzyme that brings the N4 atom of the aminopyrimidine ring to the distance required for the intramolecular C-H N hydrogen-bonding with the C2- atom of the thiazolium ring. The reactive C2 atom of TPP is the nucleophile that attacks the carbonyl carbon of different substrates used by the TPP-dependent enzymes. The structure of the heterotetrameric human pyruvate dehydrogenase (Elp) recently determined in our laboratory (1) revealed the association pattern of the subunits and the specifics of two chemically equivalent cofactor binding sites. Dynamic nonequivalence of these two cofactor sites directs the flip-flop action of this enzyme, depending upon which two active sites effect each other (2). The crystal structure derived from the holo-form of Elp provided the basis for the model of the flip-flop action of Elp in which different steps of the catalytic reaction are performed in each of the two cofactor sites at any given moment, where these steps are governed by the concerted shuttle-like motion of the subunits. It is further proposed that balancing a hydrogen-bond network and related cofactor geometry determine the continuity of catalytic events.

Effects of Alloyed Metal on the Catalysis Activity of Pt for Ethanol Partial Oxidation: Adsorption and Dehydrogenation on Pt3M (M=Pt, Ru, Sn, Re, Rh, and Pd)

PubMed Central

Xu, Zhen-Feng; Wang, Yixuan

2011-01-01

The adsorption and dehydrogenation reactions of ethanol over bimetallic clusters, Pt3M (M = Pt, Ru, Sn, Re, Rh, and Pd), have been extensively investigated with density functional theory. Both the α-hydrogen and hydroxyl adsorptions on Pt as well as on the alloyed transition metal M sites of PtM were considered as initial reaction steps. The adsorptions of ethanol on Pt and M sites of some PtM via the α-hydrogen were well established. Although the α-hydrogen adsorption on Pt site is weaker than the hydroxyl, the potential energy profiles show that the dehydrogenation via the α-hydrogen path has much lower energy barrier than that via the hydroxyl path. Generally for the α-hydrogen path the adsorption is a rate-determining-step because of rather low dehydrogenation barrier for the α-hydrogen adsorption complex (thermodynamic control), while the hydroxyl path is determined by its dehydrogenation step (kinetic control). The effects of alloyed metal on the catalysis activity of Pt for ethanol partial oxidation, including adsorption energy, energy barrier, electronic structure, and eventually rate constant were discussed. Among all of the alloyed metals only Sn enhances the rate constant of the dehydrogenation via the α-hydrogen path on the Pt site of Pt3Sn as compared with Pt alone, which interprets why the PtSn is the most active to the oxidation of ethanol. PMID:22102920

Sequence requirement of the ade6-4095 meiotic recombination hotspot in Schizosaccharomyces pombe.

PubMed

Foulis, Steven J; Fowler, Kyle R; Steiner, Walter W

2018-02-01

Homologous recombination occurs at a greatly elevated frequency in meiosis compared to mitosis and is initiated by programmed double-strand DNA breaks (DSBs). DSBs do not occur at uniform frequency throughout the genome in most organisms, but occur preferentially at a limited number of sites referred to as hotspots. The location of hotspots have been determined at nucleotide-level resolution in both the budding and fission yeasts, and while several patterns have emerged regarding preferred locations for DSB hotspots, it remains unclear why particular sites experience DSBs at much higher frequency than other sites with seemingly similar properties. Short sequence motifs, which are often sites for binding of transcription factors, are known to be responsible for a number of hotspots. In this study we identified the minimum sequence required for activity of one of such motif identified in a screen of random sequences capable of producing recombination hotspots. The experimentally determined sequence, GGTCTRGACC, closely matches the previously inferred sequence. Full hotspot activity requires an effective sequence length of 9.5 bp, whereas moderate activity requires an effective sequence length of approximately 8.2 bp and shows significant association with DSB hotspots. In combination with our previous work, this result is consistent with a large number of different sequence motifs capable of producing recombination hotspots, and supports a model in which hotspots can be rapidly regenerated by mutation as they are lost through recombination.

Utilizing hydrologic, statistical, and geochemical tools to assess uranium mobility in surface and near-surface environments

NASA Astrophysics Data System (ADS)

Naftz, D. L.; Walton-Day, K. E.; Fuller, C.; Dam, W. L.; Briggs, M. A.; Snyder, T.

2015-12-01

Legacy uranium (U) mining and processing activities have resulted in soil and water contamination on Federal, state, and tribal lands in the western United States. Sites include legacy mill sites associated with U extraction now managed by the Department of Energy and thousands of waste dumps associated with U exploration, mining, and processing. Recently (2012), over 400,000 hectares of federally managed land in northern Arizona was withdrawn from consideration of mining for a 20-year period to protect the Grand Canyon watershed from potentially adverse effects of U mineral exploration and development. Ore from active and recently active U mines in the Colorado Plateau, the Henry Mountains Complex, and the Arizona Strip is transported to the only currently (2015) active conventional mill site in the western United States, located in Utah. Previous and ongoing U.S. Geological Survey assessments to examine U mobility at a variety of legacy and active sites associated with ore exploration, extraction, and processing will be presented as field-scale examples. Topics associated with site investigations will include: (1) offsite migration of radionuclides associated with the operation of the White Mesa U mill; (2) long-term contaminant transport from legacy U waste dumps on Bureau of Land Management regulated land in Utah; (3) application of incremental soil sampling techniques to determine pre- and post-mining radionuclide levels associated with planned and operating U mines in northern Arizona; (4) application of fiber optic digital temperature sensing equipment to identify areas where shallow groundwater containing elevated U levels may be discharging to a river adjacent to a reclaimed mill site in central Wyoming; and (5) field-scale manipulation of groundwater chemistry to limit U migration from a legacy upgrader site in southeastern Utah.

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

PubMed

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

2016-01-01

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

Aryl hydrocarbon receptor-mediated activity of particulate organic matter from the Paso del Norte airshed along the U.S.-Mexico border.

PubMed Central

Arrieta, Daniel E; Ontiveros, Cynthia C; Li, Wen-Whai; Garcia, Jose H; Denison, Michael S; McDonald, Jacob D; Burchiel, Scott W; Washburn, Barbara Shayne

2003-01-01

In this study, we determined the biologic activity of dichloromethane-extracted particulate matter < 10 micro m in aerodynamic diameter (PM10) obtained from filters at three sites in the Paso del Norte airshed, which includes El Paso, Texas, USA; Juarez, Chihuahua, Mexico, and Sunland Park, New Mexico, USA. The extracts were rich in polycyclic aromatic hydrocarbons (PAHs) and had significant biologic activity, measured using two in vitro assay systems: ethoxyresorufin-(O-deethylase (EROD) induction and the aryl hydrocarbon-receptor luciferase reporter system. In most cases, both EROD (5.25 pmol/min/mg protein) and luciferase activities (994 relative light units/mg) were highest in extracts from the Advance site located in an industrial neighborhood in Juarez. These values represented 58% and 55%, respectively, of induction associated with 1 micro M ss-naphthoflavone exposures. In contrast, little activity was observed at the Northeast Clinic site in El Paso, the reference site. In most cases, luciferase and EROD activity from extracts collected from the Tillman Health Center site, situated in downtown El Paso, fell between those observed at the other two sites. Overall, a statistically significant correlation existed between PM10 and EROD and luciferase activities. Chemical analysis of extracts collected from the Advance site demonstrated that concentrations of most PAHs were higher than those reported in most other metropolitan areas in the United States. Calculations made with these data suggest a cancer risk of 5-12 cases per 100,000 people. This risk estimate, as well as comparisons with the work of other investigators, raises concern regarding the potential for adverse health effects to the residents of this airshed. Further work is needed to understand the sources, exposure, and effects of PM10 and particulate organic material in the Paso del Norte airshed. PMID:12896850

Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

DOE Office of Scientific and Technical Information (OSTI.GOV)

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, thesemore » 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.« less

Noninvasive reconstruction of the three-dimensional ventricular activation sequence during pacing and ventricular tachycardia in the rabbit heart.

PubMed

Han, Chengzong; Pogwizd, Steven M; Killingsworth, Cheryl R; He, Bin

2011-01-01

Ventricular arrhythmias represent one of leading causes for sudden cardiac death, a significant problem in public health. Noninvasive imaging of cardiac electric activities associated with ventricular arrhythmias plays an important role in better our understanding of the mechanisms and optimizing the treatment options. The present study aims to rigorously validate a novel three-dimensional (3-D) cardiac electrical imaging (3-DCEI) technique with the aid of 3-D intra-cardiac mapping during paced rhythm and ventricular tachycardia (VT) in the rabbit heart. Body surface potentials and intramural bipolar electrical recordings were simultaneously measured in a closed-chest condition in thirteen healthy rabbits. Single-site pacing and dual-site pacing were performed from ventricular walls and septum. VTs and premature ventricular complexes (PVCs) were induced by intravenous norepinephrine (NE). The non-invasively imaged activation sequence correlated well with invasively measured counterparts, with a correlation coefficient of 0.72 and a relative error of 0.30 averaged over all paced beats and NE-induced PVCs and VT beats. The averaged distance from imaged site of initial activation to measured site determined from intra-cardiac mapping was ∼5mm. These promising results suggest that 3-DCEI is feasible to non-invasively localize the origins and image activation sequence of focal ventricular arrhythmias.

Food and beverage brands that market to children and adolescents on the internet: a content analysis of branded web sites.

PubMed

Henry, Anna E; Story, Mary

2009-01-01

To identify food and beverage brand Web sites featuring designated children's areas, assess marketing techniques present on those industry Web sites, and determine nutritional quality of branded food items marketed to children. Systematic content analysis of food and beverage brand Web sites and nutrient analysis of food and beverages advertised on these Web sites. The World Wide Web. One-hundred thirty Internet Web sites of food and beverage brands with top media expenditures based on the America's Top 2000 Brands section of Brandweek magazine's annual "Superbrands" report. A standardized content analysis rating form to determine marketing techniques used on the food and beverage brand Web sites. Nutritional analysis of food brands was conducted. Of 130 Web sites analyzed, 48% featured designated children's areas. These Web sites featured a variety of Internet marketing techniques, including advergaming on 85% of the Web sites and interactive programs on 92% of the Web sites. Branded spokescharacters and tie-ins to other products were featured on the majority of the Web sites, as well. Few food brands (13%) with Web sites that market to children met the nutrition criteria set by the National Alliance for Nutrition and Activity. Nearly half of branded Web sites analyzed used designated children's areas to market food and beverages to children, 87% of which were of low nutritional quality. Nutrition professionals should advocate the use of advertising techniques to encourage healthful food choices for children.

The mode of inhibitor binding to peptidyl-tRNA hydrolase: binding studies and structure determination of unbound and bound peptidyl-tRNA hydrolase from Acinetobacter baumannii.

PubMed

Kaushik, Sanket; Singh, Nagendra; Yamini, Shavait; Singh, Avinash; Sinha, Mau; Arora, Ashish; Kaur, Punit; Sharma, Sujata; Singh, Tej P

2013-01-01

The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design.

Utilizing a Collaborative Learning Model to Promote Early Extubation Following Infant Heart Surgery.

PubMed

Mahle, William T; Nicolson, Susan C; Hollenbeck-Pringle, Danielle; Gaies, Michael G; Witte, Madolin K; Lee, Eva K; Goldsworthy, Michelle; Stark, Paul C; Burns, Kristin M; Scheurer, Mark A; Cooper, David S; Thiagarajan, Ravi; Sivarajan, V Ben; Colan, Steven D; Schamberger, Marcus S; Shekerdemian, Lara S

2016-10-01

To determine whether a collaborative learning strategy-derived clinical practice guideline can reduce the duration of endotracheal intubation following infant heart surgery. Prospective and retrospective data collected from the Pediatric Heart Network in the 12 months pre- and post-clinical practice guideline implementation at the four sites participating in the collaborative (active sites) compared with data from five Pediatric Heart Network centers not participating in collaborative learning (control sites). Ten children's hospitals. Data were collected for infants following two-index operations: 1) repair of isolated coarctation of the aorta (birth to 365 d) and 2) repair of tetralogy of Fallot (29-365 d). There were 240 subjects eligible for the clinical practice guideline at active sites and 259 subjects at control sites. Development and application of early extubation clinical practice guideline. After clinical practice guideline implementation, the rate of early extubation at active sites increased significantly from 11.7% to 66.9% (p < 0.001) with no increase in reintubation rate. The median duration of postoperative intubation among active sites decreased from 21.2 to 4.5 hours (p < 0.001). No statistically significant change in early extubation rates was found in the control sites 11.7% to 13.7% (p = 0.63). At active sites, clinical practice guideline implementation had no statistically significant impact on median ICU length of stay (71.9 hr pre- vs 69.2 hr postimplementation; p = 0.29) for the entire cohort. There was a trend toward shorter ICU length of stay in the tetralogy of Fallot subgroup (71.6 hr pre- vs 54.2 hr postimplementation, p = 0.068). A collaborative learning strategy designed clinical practice guideline significantly increased the rate of early extubation with no change in the rate of reintubation. The early extubation clinical practice guideline did not significantly change postoperative ICU length of stay.

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cooper, Jonathan B.; Weiss, Kevin L.; Coates, Leighton

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. Several active site residues in class A β-lactamases have been proposed to play key roles in monobactam hydrolysis. The protonation states of these residues have been determined previously for the apo form of a CTX-M β-lactamase. However, they have not yet been determined for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a classmore » A β-lactamase in acyl enzyme complex with aztreonam we directly observed most of the hydrogen atoms (as deuterium) within the active site in the captured acyl-enzyme state between Toho-1 β-lactamase and aztreonam. Although Lys 234 is fully protonated in the acyl-intermediate, we find that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, in agreement with previous mechanistic proposals.« less

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

DOE PAGES

Cooper, Jonathan B.; Weiss, Kevin L.; Coates, Leighton; ...

2016-10-24

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. Several active site residues in class A β-lactamases have been proposed to play key roles in monobactam hydrolysis. The protonation states of these residues have been determined previously for the apo form of a CTX-M β-lactamase. However, they have not yet been determined for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a classmore » A β-lactamase in acyl enzyme complex with aztreonam we directly observed most of the hydrogen atoms (as deuterium) within the active site in the captured acyl-enzyme state between Toho-1 β-lactamase and aztreonam. Although Lys 234 is fully protonated in the acyl-intermediate, we find that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, in agreement with previous mechanistic proposals.« less

How might the London 2012 Olympics influence health and the determinants of health? Local newspaper analysis of pre-Games pathways and impacts.

PubMed

Selvanayagam, Marinie; Thompson, Claire; Taylor, Stephanie J C; Cummins, Steven; Bourke, Liam

2012-01-01

To conduct a content analysis of pre-Games local media coverage of the potential impact on health and the determinants of health in Newham, the site of the Olympic Park. Local newspaper content analysis. Olympic park host site of the London Borough of Newham. Media coverage of employment, physical activity and well-being. Three hundred and 51 articles meeting the inclusion criteria were included in the analysis. The overwhelming majority of the articles took a positive perspective on the Olympic Games being hosted in Newham with less than 10% (32/351) addressing potential adverse effects. The frequency of articles reporting on both employment and well-being increased significantly over time (p=0.002 and p=0.006, respectively). A non-significant increasing trend was observed for physical activity (p=0.146). New employment opportunities and the promotion of physical activity in young people were the pathways most frequently reported in the local media. However, much less attention is devoted to understanding the uncertainties about how much of these new opportunities will directly improve the determinants of health in the Newham population. Pre-Games reporting on the impact on health and the determinants of health increased over time in the London Borough of Newham, and is overwhelmingly positive. However, specific uncertainties around the true nature of its impact on local employment and physical activity were articulated. Further evaluation of the tangible impacts on population health, and the determinants of health and health inequalities from the London 2012 Olympics, is required.

Long-term community dynamics of small landbirds with and without exposure to extensive disturbance from military training activities.

PubMed

Rivers, James W; Gipson, Philip S; Althoff, Donald P; Pontius, Jeffrey S

2010-02-01

Military training activities are known to impact individual species, yet our understanding of how such activities influence animal communities is limited. In this study, we used long-term data in a case study approach to examine the extent to which the local small landbird community differed between a site in northeast Kansas that experienced intensive disturbance from military training activities (Ft. Riley Military Installation) and a similar, nearby site that experienced minimal human disturbance (Konza Prairie Biological Station). In addition, we characterized how the regional pool of potential colonizers influenced local community dynamics using Breeding Bird Survey data. From 1991 to 2001, most species of small terrestrial landbirds (73%) recorded during breeding surveys were found at both sites and the mean annual richness at Ft. Riley (39.0 +/- 2.86 [SD]) was very similar to that of Konza Prairie (39.4 +/- 2.01). Richness was maintained at relatively constant levels despite compositional changes because colonizations compensated local extinctions at both sites. These dynamics were driven primarily by woodland species that exhibited stochastic losses and gains and were present at a low local and regional abundance. Our results suggest that military training activities may mimic natural disturbances for some species in this area because the small landbird community did not differ markedly between sites with and sites without extensive human disturbance. Although our results suggest that military training is not associated with large changes in the avian community, additional studies are needed to determine if this pattern is found in other ecological communities.

Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase.

PubMed

Chen, Mengbin; Chou, Wayne K W; Al-Lami, Naeemah; Faraldos, Juan A; Allemann, Rudolf K; Cane, David E; Christianson, David W

2016-05-24

Aristolochene synthase (ATAS) is a high-fidelity terpenoid cyclase that converts farnesyl diphosphate exclusively into the bicyclic hydrocarbon aristolochene. Previously determined crystal structures of ATAS complexes revealed trapped active site water molecules that could potentially interact with catalytic intermediates: water "w" hydrogen bonds with S303 and N299, water molecules "w1" and "w2" hydrogen bond with Q151, and a fourth water molecule coordinates to the Mg(2+)C ion. There is no obvious role for water in the ATAS mechanism because the enzyme exclusively generates a hydrocarbon product. Thus, these water molecules are tightly controlled so that they cannot react with carbocation intermediates. Steady-state kinetics and product distribution analyses of eight ATAS mutants designed to perturb interactions with active site water molecules (S303A, S303H, S303D, N299A, N299L, N299A/S303A, Q151H, and Q151E) indicate relatively modest effects on catalysis but significant effects on sesquiterpene product distributions. X-ray crystal structures of S303A, N299A, N299A/S303A, and Q151H mutants reveal minimal perturbation of active site solvent structure. Seven of the eight mutants generate farnesol and nerolidol, possibly resulting from addition of the Mg(2+)C-bound water molecule to the initially formed farnesyl cation, but no products are generated that would suggest enhanced reactivity of other active site water molecules. However, intermediate germacrene A tends to accumulate in these mutants. Thus, apart from the possible reactivity of Mg(2+)C-bound water, active site water molecules in ATAS are not directly involved in the chemistry of catalysis but instead contribute to the template that governs the conformation of the flexible substrate and carbocation intermediates.

Structural Analysis of Charge Discrimination in the Binding of Inhibitors to Human Carbonic Anhydrases I and II

PubMed Central

Srivastava, D. K.; Jude, Kevin M.; Banerjee, Abir L.; Haldar, Manas; Manokaran, Sumathra; Kooren, Joel; Mallik, Sanku; Christianson, David W.

2008-01-01

Despite the similarity in the active site pockets of carbonic anhydrase (CA) isozymes I and II, the binding affinities of benzenesulfonamide inhibitors are invariably higher with CA II as compared to CA I. To explore the structural basis of this molecular recognition phenomenon, we have designed and synthesized simple benzenesulfonamide inhibitors substituted at the para position with positively-charged, negatively-charged, and neutral functional groups, and we have determined the affinities and X-ray crystal structures of their enzyme complexes. The para-substituents are designed to bind in the midsection of the 15 Å deep active site cleft, where interactions with enzyme residues and solvent molecules are possible. We find that a para-substituted positively-charged amino group is more poorly tolerated in the active site of CA I compared with CA II. In contrast, a para-substituted negatively-charged carboxylate substituent is tolerated equally well in the active sites of both CA isozymes. Notably, enzyme-inhibitor affinity increases upon neutralization of inhibitor charged groups by amidation or esterification. These results inform the design of short molecular linkers connecting the benzenesulfonamide group and a para-substituted tail group in “two-prong” CA inhibitors: an optimal linker segment will be electronically neutral, yet capable of engaging in at least some hydrogen bond interactions with protein residues and/or solvent. Microcalorimetric data reveal that inhibitor binding to CA I is enthalpically less favorable and entropically more favorable than inhibitor binding to CA II. This contrasting behavior may arise in part from differences in active site desolvation and the conformational entropy of inhibitor binding to each isozyme active site. PMID:17407288

Site-Specific RNase A Activity Was Dramatically Reduced in Serum from Multiple Types of Cancer Patients

PubMed Central

Huang, Weiyan; Zhao, Mei; Wei, Na; Wang, Xiaoxia; Cao, Huqing; Du, Quan; Liang, Zicai

2014-01-01

Potent RNase activities were found in the serum of mammals but the physiological function of the RNases was never well illustrated, largely due to the caveats in methods of RNase activity measurement. None of the existing methods can distinguish between RNases with different target specificities. A systematic study was recently carried out in our lab to investigate the site-specificity of serum RNases on double-stranded RNA substrates, and found that serum RNases cleave double-stranded RNAs predominantly at 5′-U/A-3′ and 5′-C/A-3′ dinucleotide sites, in a manner closely resembling RNase A. Based on this finding, a FRET assay was developed in the current study to measure this site-specific serum RNase activity in human samples using a double stranded RNA substrate. We demonstrated that the method has a dynamic range of 10−5 mg/ml- 10−1 mg/ml using serial dilution of RNase A. The sera of 303 cancer patients were subjected to comparison with 128 healthy controls, and it was found that serum RNase activities visualized with this site-specific double stranded probe were found to be significantly reduced in patients with gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, ovary cancer, cervical cancer, bladder cancer, kidney cancer and lung cancer, while only minor changes were found in breast and colon cancer patients. This is the first report using double stranded RNA as probe to quantify site-specific activities of RNase A in a serum. The results illustrated that RNase A might be further evaluated to determine if it can serve as a new class of biomarkers for certain cancer types. PMID:24805924

Social support for physical activity-role of Facebook with and without structured intervention.

PubMed

Cavallo, David N; Tate, Deborah F; Ward, Dianne S; DeVellis, Robert F; Thayer, Linden M; Ammerman, Alice S

2014-12-01

Despite their widespread use and extensive technical features, little is known about how to use online social networking sites to increase physical activity. This study aims to examine Facebook engagement among participants in the online social networking arm of a randomized controlled physical activity promotion trial (n = 67). Facebook communications were double coded and analyzed using ATLAS.ti. Regression procedures were used to determine predictors of Facebook use and associations between types of use and changes in perceived social support and physical activity. Changes in perceived social support and physical activity were more strongly associated with participants' individual Facebook use than use of the Facebook intervention group. The way social media sites are used in intervention design could have an impact on their effects. Including existing friends in interventions and using applications that incorporate intervention activities into a more naturalistic use of Facebook may improve the efficacy of future interventions.

Chemical structure determines target organ carcinogenesis in rats

PubMed Central

Carrasquer, C. A.; Malik, N.; States, G.; Qamar, S.; Cunningham, S.L.; Cunningham, A.R.

2012-01-01

SAR models were developed for 12 rat tumour sites using data derived from the Carcinogenic Potency Database. Essentially, the models fall into two categories: Target Site Carcinogen – Non-Carcinogen (TSC-NC) and Target Site Carcinogen – Non-Target Site Carcinogen (TSC-NTSC). The TSC-NC models were composed of active chemicals that were carcinogenic to a specific target site and inactive ones that were whole animal non-carcinogens. On the other hand, the TSC-NTSC models used an inactive category also composed of carcinogens but to any/all other sites but the target site. Leave one out validations produced an overall average concordance value for all 12 models of 0.77 for the TSC-NC models and 0.73 for the TSC-NTSC models. Overall, these findings suggest that while the TSC-NC models are able to distinguish between carcinogens and non-carcinogens, the TSC-NTSC models are identifying structural attributes that associate carcinogens to specific tumour sites. Since the TSC-NTSC models are composed of active and inactive compounds that are genotoxic and non-genotoxic carcinogens, the TSC-NTSC models may be capable of deciphering non-genotoxic mechanisms of carcinogenesis. Together, models of this type may also prove useful in anticancer drug development since they essentially contain chemicals moieties that target specific tumour site. PMID:23066888

Assessment of trace element contamination of urban surface soil at informal industrial sites in a low-income country.

PubMed

Kanda, Artwell; Ncube, France; Hwende, Tamuka; Makumbe, Peter

2018-05-29

Trace elements released by human activity are ubiquitously detected in surface soil. The trace element contamination statuses of 20 sampling stations at two busy informal industrial sites of Harare city, Zimbabwe, were evaluated using geochemical indices. Spectrophotometric determinations of concentrations of trace elements in surface soil indicated generally higher values than the reference site and the average upper earth's crust. High contamination factors were observed for trace elements across sampling stations at Gazaland and Siyaso informal industrial sites. Concentrations exhibited heterogeneous distribution of trace elements in surface soil varying with the nature of activity at a sampling station. The pollution load index and degree of contamination suggested highly contaminated surface soil with Cd, Cu and Pb particularly where the following activities were done: (1) welding, (2) automobile maintenance and (3) waste dumping. These results may be very important to reduce soil contamination. Paving surfaces may help to reduce dispersal of trace elements deposited on surface soil to other stations and minimise human exposure via inhalation and contact.

Structure of Bacillus subtilis γ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ida, Tomoyo; Suzuki, Hideyuki; Fukuyama, Keiichi

2014-02-01

The binding modes of acivicin, a classical and an electrophilic active-site-directed glutamate analogue, to bacterial γ-glutamyltranspeptidases were found to be diverse. γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of acivicin bound to Bacillus subtilis GGT determined by X-ray crystallography to 1.8 Å resolution is presented, in which it binds to the active site in a similar manner to that in Helicobacter pylori GGT, but in a different binding mode to that in Escherichia coli GGT. In B. subtilis GGT, acivicin is bound covalentlymore » through its C3 atom with sp{sup 2} hybridization to Thr403 O{sup γ}, the catalytic nucleophile of the enzyme. The results show that acivicin-binding sites are common, but the binding manners and orientations of its five-membered dihydroisoxazole ring are diverse in the binding pockets of GGTs.« less

Crystallographic snapshots of active site metal shift in E. coli fructose 1,6-bisphosphate aldolase.

PubMed

Tran, Huyen-Thi; Lee, Seon-Hwa; Ho, Thien-Hoang; Hong, Seung-Hye; Huynh, Kim-Hung; Ahn, Yeh-Jin; Oh, Deok-Kun; Kang, Lin-Woo

2016-12-01

Fructose 1,6-bisphosphate aldolase (FBA) is important for both glycolysis and gluconeogenesis in life. Class II (zinc dependent) FBA is an attractive target for the development of antibiotics against protozoa, bacteria, and fungi, and is also widely used to produce various high-value stereoisomers in the chemical and pharmaceutical industry. In this study, the crystal structures of class II Escherichia coli FBA (EcFBA) were determined from four different crystals, with resolutions between 1.8 Å and 2.0 Å. Native EcFBA structures showed two separate sites of Zn1 (interior position) and Zn2 (active site surface position) for Zn2＋ ion. Citrate and TRIS bound EcFBA structures showed Zn2＋ position exclusively at Zn2. Crystallographic snapshots of EcFBA structures with and without ligand binding proposed the rationale of metal shift at the active site, which might be a hidden mechanism to keep the trace metal cofactor Zn2＋ within EcFBA without losing it. [BMB Reports 2016; 49(12): 681-686].

Identification of Bacteria Synthesizing Ribosomal RNA in Response to Uranium Addition During Biostimulation at the Rifle, CO Integrated Field Research Site

PubMed Central

McGuinness, Lora R.; Wilkins, Michael J.; Williams, Kenneth H.; Long, Philip E.; Kerkhof, Lee J.

2015-01-01

Understanding which organisms are capable of reducing uranium at historically contaminated sites provides crucial information needed to evaluate treatment options and outcomes. One approach is determination of the bacteria which directly respond to uranium addition. In this study, uranium amendments were made to groundwater samples from a site of ongoing biostimulation with acetate. The active microbes in the planktonic phase were deduced by monitoring ribosomes production via RT-PCR. The results indicated several microorganisms were synthesizing ribosomes in proportion with uranium amendment up to 2 μM. Concentrations of U (VI) >2 μM were generally found to inhibit ribosome synthesis. Two active bacteria responding to uranium addition in the field were close relatives of Desulfobacter postgateii and Geobacter bemidjiensis. Since RNA content often increases with growth rate, our findings suggest it is possible to rapidly elucidate active bacteria responding to the addition of uranium in field samples and provides a more targeted approach to stimulate specific populations to enhance radionuclide reduction in contaminated sites. PMID:26382047

Effects of subsoiling on lateral roots, sucrose metabolizing enzymes, and soil ergosterol in two Jeffrey pine stands

Treesearch

W.J. Otrosina; Shi-Jean S. Sung; L.M. White

1996-01-01

We determined the effects of subsoiling on woody lateral roots and enzyme activities involved in stem carbon metabolism of 90- to 100-year-old Jeffrey pine (Pinus jeffreyi Grev. And Balf.) growing on the eastern side of the California Sierra Nevada Range.Twelve 1.0-ha plots were established on each of two sites. Four site treatments thinning and subsoiling entire...

Why Does a Large Prison Population Yield So Few Participants in a College Program Offered at Prison Sites?

ERIC Educational Resources Information Center

Walsh, S. M. Steve

A study was conducted to determine why such a small number (less than 2 percent) of the approximately 9,000 adult male prisoners housed in the 3 complexes of the California Institution for Men (Chino, California) in the mid-1980s were actively participating in the college program offered at the prison sites. Data were collected through interviews…

Aquatic disposal field investigations Galveston, Texas, offshore disposal site. Evaluative summary. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wright, T.D.; Mathis, D.B.; Brannon, J.M.

This study was part of an investigation to determine the environmental effects of offshore dredged material disposal at Galveston, Texas. The biological portion of the study was conducted in two phases: a pilot survey of the dredged material disposal site (DMDS) to determine the areal distribution of the biota and sediments; and an experimental study to assess the effect of dredged material disposal on the biota at selected sites in the DMDS. Three experimental sites were investigated: a sandy bottom that received sand, shell, and silt-clay dredged material; a muddy bottom that received sand and shell dredged material; and amore » muddy bottom that received silt-clay dredged material. The magnitude of the effect on the benthic populations could not be accurately assessed because adequate predisposal data on natural sediment and benthic population changes were not available. Dredged material deposits had no apparent effect on feeding habits of fish or on the distribution of nekton, although some nektonic species may have congregated in the turbid water following dredged material disposal. Zooplankton and phytoplankton studies detected no population changes during disposal that could not have been due to sampling error. It is probable that sudden abiotic changes and commercial fishing activities cause more destruction of biota than dredging-related activities.« less

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation.

PubMed

Cho, Seong-Jun; Kang, Hana; Kim, Min Young; Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun; Pyo, Suhkneung; Yang, Kwang Hee

2016-04-01

To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Splenocytes and IM-9 cells were uniformly irradiated with various doses of a (137)Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast. Copyright © 2016 Elsevier Inc. All rights reserved.

Memory, reconsolidation and extinction in Lymnaea require the soma of RPeD1.

PubMed

Sangha, Susan; Varshney, Nishi; Fras, Mary; Smyth, Kim; Rosenegger, David; Parvez, Kashif; Sadamoto, Hisayo; Lukowiak, Ken

2004-01-01

The central pattern generator (CPG) that drives aerial respiratory behaviour in Lymnaea consists of 3 neurons. One of these, RPeD1--the cell that initiates activity in the circuit, plays an absolutely necessary role as a site for memory formation, memory reconsolidation, and extinction. Using an operant conditioning training procedure that results in a long-term non-declarative memory (LTM), we decrease the occurrence of aerial respiratory behaviour. Since snails can still breathe cutaneously learning this procedure is not harmful. Concomitant with behavioural memory are changes in the spiking activity of RPeD1. Going beyond neural correlates of memory we directly show that RPeD1 is a necessary site for LTM formation. Expanding on this finding we show that this neuron is also a necessary site for memory reconsolidation and 'Pavlovian' extinction. As far as we can determine, this is the first time a single neuron has been shown to be a necessary site for these different aspects memory. RPeD1 is thus a key neuron mediating different hierarchical aspects of memory. We are now in a position to determine the necessary neuronal, molecular and proteomic events in this neuron that are causal to memory formation, reconsolidation and extinction.

Trimethoprim in vitro antibacterial activity is not increased by adding sulfamethoxazole for pediatric Escherichia coli urinary tract infection.

PubMed

Nguyen, Hiep T; Hurwitz, Richard S; Defoor, W Robert; Minevich, Eugene; McAdam, Alexander J; Mortensen, Joel E; Novak-Weekley, Susan M; Minnillo, Brian J; Elder, Jack S

2010-07-01

The combination of trimethoprim/sulfamethoxazole is often used to treat uncomplicated urinary tract infections in children. The rationale for combining trimethoprim and sulfamethoxazole is that they may act synergistically to increase antibacterial activity. However, approximately 3% of patients show allergic reactions to sulfamethoxazole, of which some are serious (liver failure and Stevens-Johnson syndrome). We determined whether adding sulfamethoxazole is necessary to increase in vitro antibacterial activity for pediatric urinary tract infection compared to that of trimethoprim alone. We prospectively identified 1,298 children with urinary tract infection (greater than 100,000 cfu/ml Escherichia coli) from a total of 4 American regions. In vitro susceptibility of bacterial isolates to sulfamethoxazole, trimethoprim and trimethoprim/sulfamethoxazole was determined using disk diffusion. Ampicillin susceptibility was tested at 2 sites. At 1 site all uropathogens from consecutive urinary isolates were evaluated. E. coli susceptibility to trimethoprim was 70%, comparable to the 70% of trimethoprim/sulfamethoxazole (p = 0.9) and higher than the 56.9% of sulfamethoxazole (p <0.05). This susceptibility pattern was without regional differences. At 2 sites susceptibility to trimethoprim was significantly higher than to ampicillin. At 1 site the susceptibility of other uropathogens to trimethoprim and trimethoprim/sulfamethoxazole was similar to that of E. coli. In children with urinary tract infection in vitro susceptibility to trimethoprim was comparable to that to trimethoprim/sulfamethoxazole and significantly higher than to sulfamethoxazole. This finding was similar at all sites. Adding sulfamethoxazole appears unnecessary and may represent a risk to patients. Trimethoprim can be used as an alternative to trimethoprim/sulfamethoxazole based on in vitro antibacterial susceptibility. Routine trimethoprim/sulfamethoxazole use for urinary tract infection should be carefully reevaluated. Copyright (c) 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Single-fluorophore monitoring of DNA hybridization for investigating the effect of secondary structure on the nucleation step.

PubMed

Jo, Joon-Jung; Kim, Min-Ji; Son, Jung-Tae; Kim, Jandi; Shin, Jong-Shik

2009-07-17

Nucleic acid hybridization is one of the essential biological processes involved in storage and transmission of genetic information. Here we quantitatively determined the effect of secondary structure on the hybridization activation energy using structurally defined oligonucleotides. It turned out that activation energy is linearly proportional to the length of a single-stranded region flanking a nucleation site, generating a 0.18 kcal/mol energy barrier per nucleotide. Based on this result, we propose that the presence of single-stranded segments available for non-productive base pairing with a nucleation counterpart extends the searching process for nucleation sites to find a perfect match. This result may provide insights into rational selection of a target mRNA site for siRNA and antisense gene silencing.

Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits myeloperoxidase activity in inflammatory cells.

PubMed

Mishra, Om P; Popov, Anatoliy V; Pietrofesa, Ralph A; Nakamaru-Ogiso, Eiko; Andrake, Mark; Christofidou-Solomidou, Melpo

2018-06-01

Myeloperoxidase (MPO) generates hypochlorous acid (HOCl) during inflammation and infection. We showed that secoisolariciresinol diglucoside (SDG) scavenges radiation-induced HOCl in physiological solutions. However, the action of SDG and its synthetic version, LGM2605, on MPO-catalyzed generation of HOCl is unknown. The present study evaluated the effect of LGM2605 on human MPO, and murine MPO from macrophages and neutrophils. MPO activity was determined fluorometrically using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF). The effect of LGM2605 on (a) the peroxidase cycle of MPO was determined using Amplex Red while the effect on (b) the chlorination cycle was determined using a taurine chloramine assay. Using electron paramagnetic resonance (EPR) spectroscopy we determined the effect of LGM2605 on the EPR signals of MPO. Finally, computational docking of SDG was used to identify energetically favorable docking poses to enzyme's active site. LGM2605 inhibited human and murine MPO activity. MPO inhibition was observed in the absence and presence of Cl - . EPR confirmed that LGM2605 suppressed the formation of Compound I, an oxoiron (IV) intermediate [Fe(IV)O] containing a porphyrin π-radical of MPO's catalytic cycle. Computational docking revealed that SDG can act as an inhibitor by binding to the enzyme's active site. We conclude that LGM2605 inhibits MPO activity by suppressing both the peroxidase and chlorination cycles. EPR analysis demonstrated that LGM2605 inhibits MPO by decreasing the formation of the highly oxidative Compound I. This study identifies a novel mechanism of LGM2605 action as an inhibitor of MPO and indicates that LGM2605 may be a promising attenuator of oxidant-dependent inflammatory tissue damage. Copyright © 2018 Elsevier B.V. All rights reserved.

Control of peroxisome proliferator-activated receptor gamma2 stability and activity by SUMOylation.

PubMed

Floyd, Z Elizabeth; Stephens, Jacqueline M

2004-06-01

To determine whether small ubiquitin-related modifier (SUMO)ylation of lysine 107 plays a role in regulating the activity of peroxisome proliferator-activated receptor gamma (PPARgamma). Transient expression of wild-type and K107R-PPARgamma2 in the NIH 3T3 fibroblast cell line was carried out in conjunction with half-life studies, luciferase activity assays, and indirect immunofluorescence localization studies. Additional in vitro analysis was carried out using recombinant SUMOylation pathway proteins along with in vitro transcribed and translated wild-type or K107R-PPARgamma2 to examine the SUMO-1 modification state of wild-type and SUMO-deficient K107R-PPARgamma2. While examining PPARgamma2 for potential ubiquitylation sites, we identified a strong consensus site for SUMO modification that contains lysine 107. In vitro, SUMOylation studies showed that lysine 107 of PPARgamma2 is a major SUMOylation site and that at least one other SUMOylation site is present in PPARgamma. In addition, our results demonstrated that SUMO-1 affects PPARgamma stability and transcriptional activity but not the nuclear localization of PPARgamma. These results indicated that SUMOylation plays a role in regulating PPARgamma, both indirectly and directly by modification of lysine 107. Because PPARgamma is regulated in numerous animal models of obesity, understanding the covalent modifications of PPARgamma may enhance our understanding of the metabolic syndrome.

Cortico-cortical evoked potentials for sites of early versus late seizure spread in stereoelectroencephalography.

PubMed

Lega, Bradley; Dionisio, Sasha; Flanigan, Patrick; Bingaman, William; Najm, Imad; Nair, Dileep; Gonzalez-Martinez, Jorge

2015-09-01

Cortico-cortical evoked potentials offer the possibility of understanding connectivity within seizure networks to improve diagnosis and more accurately identify candidates for seizure surgery. We sought to determine if cortico-cortical evoked potentials and post-stimulation oscillatory changes differ for sites of EARLY versus LATE ictal spread. 37 patients undergoing stereoelectroencephalography were tested using a cortico-cortical evoked potential paradigm. All electrodes were classified according to the speed of ictal spread. EARLY spread sites were matched to a LATE spread site equidistant from the onset zone. Root-mean-square was used to quantify evoked responses and post-stimulation gamma band power and coherence were extracted and compared. Sites of EARLY spread exhibited significantly greater evoked responses after stimulation across all patients (t(36)=2.973, p=0.004). Stimulation elicited enhanced gamma band activity at EARLY spread sites (t(36)=2.61, p=0.03, FDR corrected); this gamma band oscillation was highly coherent with the onset zone. Cortico-cortical evoked potentials and post-stimulation changes in gamma band activity differ between sites of EARLY versus LATE ictal spread. The oscillatory changes can help visualize connectivity within the seizure network. Copyright © 2015 Elsevier B.V. All rights reserved.

Dynamic asymmetry and the role of the conserved active-site thiol in rabbit muscle creatine kinase.

PubMed

Londergan, Casey H; Baskin, Rachel; Bischak, Connor G; Hoffman, Kevin W; Snead, David M; Reynoso, Christopher

2015-01-13

Symmetric and asymmetric crystal structures of the apo and transition state analogue forms, respectively, of the dimeric rabbit muscle creatine kinase have invoked an "induced fit" explanation for asymmetry between the two subunits and their active sites. However, previously reported thiol reactivity studies at the dual active-site cysteine 283 residues suggest a more latent asymmetry between the two subunits. The role of that highly conserved active-site cysteine has also not been clearly determined. In this work, the S-H vibrations of Cys283 were observed in the unmodified MM isoform enzyme via Raman scattering, and then one and both Cys283 residues in the same dimeric enzyme were modified to covalently attach a cyano group that reports on the active-site environment via its infrared CN stretching absorption band while maintaining the catalytic activity of the enzyme. Unmodified and Cys283-modified enzymes were investigated in the apo and transition state analogue forms of the enzyme. The narrow and invariant S-H vibrational bands report a homogeneous environment for the unmodified active-site cysteines, indicating that their thiols are hydrogen bonded to the same H-bond acceptor in the presence and absence of the substrate. The S-H peak persists at all physiologically relevant pH's, indicating that Cys283 is protonated at all pH's relevant to enzymatic activity. Molecular dynamics simulations identify the S-H hydrogen bond acceptor as a single, long-resident water molecule and suggest that the role of the conserved yet catalytically unnecessary thiol may be to dynamically rigidify that part of the active site through specific H-bonding to water. The asymmetric and broad CN stretching bands from the CN-modified Cys283 suggest an asymmetric structure in the apo form of the enzyme in which there is a dynamic exchange between spectral subpopulations associated with water-exposed and water-excluded probe environments. Molecular dynamics simulations indicate a homogeneous orientation of the SCN probe group in the active site and thus rule out a local conformational explanation at the residue level for the multipopulation CN stretching bands. The homogeneous simulated SCN orientation suggests strongly that a more global asymmetry between the two subunits is the cause of the CN probe's broad and asymmetric infrared line shape. Together, these spectral observations localized at the active-site cysteines indicate an intrinsic, dynamic asymmetry between the two subunits that exists already in the apo form of the dimeric creatine kinase enzyme, rather than being induced by the substrate. Biochemical and methodological consequences of these conclusions are considered.

Changes in recreation values after fire in the northern Rocky Mountains

Treesearch

Flowers. Patrick J.; Henry J. Vaux; Philip D. Gardner; Thomas J. Mills

1985-01-01

Changes in recreation values after wildfire in the northern Rocky Mountains were determined by estimating the difference in the present net value of recreation activity with and without fire. To estimate the value of recreation activity at burned and unburned sites, a contingent market valuation approach was used. Hypothetical market transactions were created by...

Engagement with Online Self-Tests as a Predictor of Student Success

ERIC Educational Resources Information Center

Thomas, Judith A.; Wadsworth, Dan; Jin, Ying; Clarke, Jim; Page, Rachel; Thunders, Michelle

2017-01-01

Online self-testing as part of the online learning environment (OLE) provides practice questions on key concepts with immediate feedback--in a "no-risk" environment. OLE activity was analysed for 471 on-site and distance students enrolled in health science courses to determine total activity on the OLE and usage of online self-tests. The…

76 FR 78708 - Self-Regulatory Organizations; Chicago Board Options Exchange, Incorporated; Notice of Filing and...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-19

... the Exchange's Web site ( http://www.cboe.org/Legal ), at the Exchange's Office of the Secretary and... COB (referred to herein as the ``re-COA'' feature), which is currently in use but not expressly... is activated, the Exchange may also determine to activate the re-COA feature for complex orders...

How Accurate Is Your Activity Tracker? A Comparative Study of Step Counts in Low-Intensity Physical Activities

PubMed Central

2017-01-01

Background As commercially available activity trackers are being utilized in clinical trials, the research community remains uncertain about reliability of the trackers, particularly in studies that involve walking aids and low-intensity activities. While these trackers have been tested for reliability during walking and running activities, there has been limited research on validating them during low-intensity activities and walking with assistive tools. Objective The aim of this study was to (1) determine the accuracy of 3 Fitbit devices (ie, Zip, One, and Flex) at different wearing positions (ie, pants pocket, chest, and wrist) during walking at 3 different speeds, 2.5, 5, and 8 km/h, performed by healthy adults on a treadmill; (2) determine the accuracy of the mentioned trackers worn at different sites during activities of daily living; and (3) examine whether intensity of physical activity (PA) impacts the choice of optimal wearing site of the tracker. Methods We recruited 15 healthy young adults to perform 6 PAs while wearing 3 Fitbit devices (ie, Zip, One, and Flex) on their chest, pants pocket, and wrist. The activities include walking at 2.5, 5, and 8 km/h, pushing a shopping cart, walking with aid of a walker, and eating while sitting. We compared the number of steps counted by each tracker with gold standard numbers. We performed multiple statistical analyses to compute descriptive statistics (ie, ANOVA test), intraclass correlation coefficient (ICC), mean absolute error rate, and correlation by comparing the tracker-recorded data with that of the gold standard. Results All the 3 trackers demonstrated good-to-excellent (ICC>0.75) correlation with the gold standard step counts during treadmill experiments. The correlation was poor (ICC<0.60), and the error rate was significantly higher in walker experiment compared to other activities. There was no significant difference between the trackers and the gold standard in the shopping cart experiment. The wrist worn tracker, Flex, counted several steps when eating (P<.01). The chest tracker was identified as the most promising site to capture steps in more intense activities, while the wrist was the optimal wearing site in less intense activities. Conclusions This feasibility study focused on 6 PAs and demonstrated that Fitbit trackers were most accurate when walking on a treadmill and least accurate during walking with a walking aid and for low-intensity activities. This may suggest excluding participants with assistive devices from studies that focus on PA interventions using commercially available trackers. This study also indicates that the wearing site of the tracker is an important factor impacting the accuracy performance. A larger scale study with a more diverse population, various activity tracker vendors, and a larger activity set are warranted to generalize our results. PMID:28801304

Structural mechanism of enoyl-CoA hydratase: three atoms from a single water are added in either an E1cb stepwise or concerted fashion.

PubMed

Bahnson, Brian J; Anderson, Vernon E; Petsko, Gregory A

2002-02-26

We have determined the crystal structure of the enzyme enoyl-CoA hydratase (ECH) from rat liver with the bound substrate 4-(N,N-dimethylamino)cinnamoyl-CoA using X-ray diffraction data to a resolution of 2.3 A. In addition to the thiolester substrate, the catalytic water, which is added in the hydration reaction, has been modeled into well-defined electron density in each of the six active sites of the physiological hexamer within the crystallographic asymmetric unit. The catalytic water bridges Glu(144) and Glu(164) of the enzyme and has a lone pair of electrons poised to react with C(3) of the enzyme-bound alpha,beta-unsaturated thiolester. The water molecule, which bridges two glutamate residues, is reminiscent of the enolase active site. However, unlike enolase, which has a lysine available to donate a proton, there are no other sources of protons available from other active site residues in ECH. Furthermore, an analysis of the hydrogen-bonding network of the active site suggests that both Glu(144) and Glu(164) are ionized and carry a negative charge with no reasonable place to have a protonated carboxylate. This lack of hydrogen-bonding acceptors that could accommodate a source of a proton, other than from the water molecule, leads to a hypothesis that the three atoms from a single water molecule are added across the double bond to form the hydrated product. The structural results are discussed in connection with details of the mechanism, which have been elucidated from kinetics, site-directed mutagenesis, and spectroscopy of enzyme-substrate species, in presenting an atomic-resolution mechanism of the reaction. Contrary to the previous interpretation, the structure of the E-S complex together with previously determined kinetic isotope effects is consistent with either a concerted mechanism or an E1cb stepwise mechanism.

Chemical rescue of the post-translationally carboxylated lysine mutant of allantoinase and dihydroorotase by metal ions and short-chain carboxylic acids.

PubMed

Ho, Ya-Yeh; Huang, Yen-Hua; Huang, Cheng-Yang

2013-04-01

Bacterial allantoinase (ALLase) and dihydroorotase (DHOase) are members of the cyclic amidohydrolase family. ALLase and DHOase possess similar binuclear metal centers in the active site in which two metals are bridged by a post-translationally carboxylated lysine. In this study, we determined the effects of carboxylated lysine and metal binding on the activities of ALLase and DHOase. Although DHOase is a metalloenzyme, purified DHOase showed high activity without additional metal supplementation in a reaction mixture or bacterial culture. However, unlike DHOase, ALLase had no activity unless some specific metal ions were added to the reaction mixture or culture. Substituting the metal binding sites H59, H61, K146, H186, H242, or D315 with alanine completely abolished the activity of ALLase. However, the K146C, K146D and K146E mutants of ALLase were still active with about 1-6% activity of the wild-type enzyme. These ALLase K146 mutants were found to have 1.4-1.7 mol metal per mole enzyme subunit, which may indicate that they still contained the binuclear metal center in the active site. The activity of the K146A mutant of the ALLase and the K103A mutant of DHOase can be chemically rescued by short-chain carboxylic acids, such as acetic, propionic, and butyric acids, but not by ethanol, propan-1-ol, and imidazole, in the presence of Co2+ or Mn2+ ions. However, the activity was still ~10-fold less than that of wild-type ALLase. Overall, these results indicated that the 20 natural basic amino acid residues were not sufficiently able to play the role of lysine. Accordingly, we proposed that during evolution, the post-translational modification of carboxylated lysine in the cyclic amidohydrolase family was selected for promoting binuclear metal center self-assembly and increasing the nucleophilicity of the hydroxide at the active site for enzyme catalysis. This kind of chemical rescue combined with site-directed mutagenesis may also be used to identify a binuclear metal center in the active site for other metalloenzymes.

Structure, function and regulation of the enzymes in the starch biosynthetic pathway.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geiger, Jim

Starch is the major reserve polysaccharide in nature and accounts for the majority of the caloric intact of humans. It is also gaining importance as a renewable and biodegradable industrial material. There is burgeoning interest in increasing the amount and altering the properties of the plant starches by plant genetic modification. A rational approach to this effort will require a detailed, atomic-level understanding of the enzymatic processes that produce the starch granule. The starch granule is a complex particle made up of alternating layers of crystalline and amorphous lamellae. It consists of two types of polymer, amylose, a polymer ofmore » relatively long chains of α-1,4-linked glucans that contain virtually no branches, and amylopectin, which is highly branched and contains much shorter chains. This complex structure is synthesized by the coordinate activities of the starch synthases (SS), which elongate the polysaccharide chain by addition of glucose units via α-1,4 linkages using ADP- glucose as a donor, and branching enzymes (BE), which branch the polysaccharide chain by cleavage of α₋1,4 linkages and subsequent re-attachment via α₋1,6 linkages. Several isoforms of both starch synthase (SS) and branching enzyme (BE) are found in plants, including SSI, SSII, SSIII and granule- bound SS (GBSS), and SBEI, SBEIIa and SBEIIb. These isoforms have different activities and substrate and product specificities and play different roles in creating the granule and determining the properties of the resulting starch. The overarching goal of this proposal is to begin to understand the regulation and specificities of these enzymes at the atomic level. High-resolution X-ray structures of these enzymes bound to substrates and products will be determined to visualize the molecular interactions responsible for the properties of the enzymes. Hypotheses regarding these issues will then be tested using mutagenesis and enzyme assays. To date, we have determined the structure of ADP- Glucose pyrophosphorylase from potato in its inhibited conformation, and bound to both ATP and ADP-glucose. In addition, we have determined the first structure of glycogen synthase in its "closed", catalytically active conformation bound to ADP-glucose. We also determined the structure of glycogen synthase bound to malto-oligosaccharides, showing for the first time that an enzyme in the starch biosynthetic pathway recognizes glucans not just in its active site but on binding sites on the surface of the enzyme ten’s of Angstroms from the active site. In addition our structure of a glycogen branching enzyme bound to malto-oligosaccharides identified seven distinct binding sites distributed about the surface of the enzyme. We will now determine the function of these sites to get a molecular-level picture of exactly how these enzymes interact with their polymeric substrates and confer specificity leading to the complex structure of the starch granule. We will extend our studies to other isoforms of the enzymes, to understand how their structures give rise to their distinct function. Our goal is to understand what accounts for the various functional differences between SS and SBE isoforms at a molecular level.« less

Sequence features of viral and human Internal Ribosome Entry Sites predictive of their activity

PubMed Central

Elias-Kirma, Shani; Nir, Ronit; Segal, Eran

2017-01-01

Translation of mRNAs through Internal Ribosome Entry Sites (IRESs) has emerged as a prominent mechanism of cellular and viral initiation. It supports cap-independent translation of select cellular genes under normal conditions, and in conditions when cap-dependent translation is inhibited. IRES structure and sequence are believed to be involved in this process. However due to the small number of IRESs known, there have been no systematic investigations of the determinants of IRES activity. With the recent discovery of thousands of novel IRESs in human and viruses, the next challenge is to decipher the sequence determinants of IRES activity. We present the first in-depth computational analysis of a large body of IRESs, exploring RNA sequence features predictive of IRES activity. We identified predictive k-mer features resembling IRES trans-acting factor (ITAF) binding motifs across human and viral IRESs, and found that their effect on expression depends on their sequence, number and position. Our results also suggest that the architecture of retroviral IRESs differs from that of other viruses, presumably due to their exposure to the nuclear environment. Finally, we measured IRES activity of synthetically designed sequences to confirm our prediction of increasing activity as a function of the number of short IRES elements. PMID:28922394

Migrant Education Administrative Guide.

ERIC Educational Resources Information Center

North Carolina State Dept. of Public Instruction, Raleigh. Div. of Compensatory Education.

Relating specifically to the North Carolina migrant education program's administrative responsibilities, this guide is designed to aid administrators in program management, monitoring project activities, project evaluation, self-assessment, determining needs for training and staff development, site-visit preparation, policy development, and…

Crystal structures of C4 form maize and quaternary complex of E. coli phosphoenolpyruvate carboxylases.

PubMed

Matsumura, Hiroyoshi; Xie, Yong; Shirakata, Shunsuke; Inoue, Tsuyoshi; Yoshinaga, Takeo; Ueno, Yoshihisa; Izui, Katsura; Kai, Yasushi

2002-12-01

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the first step in the fixation of atmospheric CO(2) during C(4) photosynthesis. The crystal structure of C(4) form maize PEPC (ZmPEPC), the first structure of the plant PEPCs, has been determined at 3.0 A resolution. The structure includes a sulfate ion at the plausible binding site of an allosteric activator, glucose 6-phosphate. The crystal structure of E. coli PEPC (EcPEPC) complexed with Mn(2+), phosphoenolpyruvate analog (3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate), and an allosteric inhibitor, aspartate, has also been determined at 2.35 A resolution. Dynamic movements were found in the ZmPEPC structure, compared with the EcPEPC structure, around two loops near the active site. On the basis of these molecular structures, the mechanisms for the carboxylation reaction and for the allosteric regulation of PEPC are proposed.

Tumor suppressor genes are larger than apoptosis-effector genes and have more regions of active chromatin: Connection to a stochastic paradigm for sequential gene expression programs.

PubMed

Garcia, Marlene; Mauro, James A; Ramsamooj, Michael; Blanck, George

2015-08-03

Apoptosis- and proliferation-effector genes are substantially regulated by the same transactivators, with E2F-1 and Oct-1 being notable examples. The larger proliferation-effector genes have more binding sites for the transactivators that regulate both sets of genes, and proliferation-effector genes have more regions of active chromatin, i.e, DNase I hypersensitive and histone 3, lysine-4 trimethylation sites. Thus, the size differences between the 2 classes of genes suggest a transcriptional regulation paradigm whereby the accumulation of transcription factors that regulate both sets of genes, merely as an aspect of stochastic behavior, accumulate first on the larger proliferation-effector gene "traps," and then accumulate on the apoptosis effector genes, thereby effecting sequential activation of the 2 different gene sets. As IRF-1 and p53 levels increase, tumor suppressor proteins are first activated, followed by the activation of apoptosis-effector genes, for example during S-phase pausing for DNA repair. Tumor suppressor genes are larger than apoptosis-effector genes and have more IRF-1 and p53 binding sites, thereby likewise suggesting a paradigm for transcription sequencing based on stochastic interactions of transcription factors with different gene classes. In this report, using the ENCODE database, we determined that tumor suppressor genes have a greater number of open chromatin regions and histone 3 lysine-4 trimethylation sites, consistent with the idea that a larger gene size can facilitate earlier transcriptional activation via the inclusion of more transactivator binding sites.

Crystal structure of the dithiol oxidase DsbA enzyme from proteus mirabilis bound non-covalently to an active site peptide ligand.

PubMed

Kurth, Fabian; Duprez, Wilko; Premkumar, Lakshmanane; Schembri, Mark A; Fairlie, David P; Martin, Jennifer L

2014-07-11

The disulfide bond forming DsbA enzymes and their DsbB interaction partners are attractive targets for development of antivirulence drugs because both are essential for virulence factor assembly in Gram-negative pathogens. Here we characterize PmDsbA from Proteus mirabilis, a bacterial pathogen increasingly associated with multidrug resistance. PmDsbA exhibits the characteristic properties of a DsbA, including an oxidizing potential, destabilizing disulfide, acidic active site cysteine, and dithiol oxidase catalytic activity. We evaluated a peptide, PWATCDS, derived from the partner protein DsbB and showed by thermal shift and isothermal titration calorimetry that it binds to PmDsbA. The crystal structures of PmDsbA, and the active site variant PmDsbAC30S were determined to high resolution. Analysis of these structures allows categorization of PmDsbA into the DsbA class exemplified by the archetypal Escherichia coli DsbA enzyme. We also present a crystal structure of PmDsbAC30S in complex with the peptide PWATCDS. The structure shows that the peptide binds non-covalently to the active site CXXC motif, the cis-Pro loop, and the hydrophobic groove adjacent to the active site of the enzyme. This high-resolution structural data provides a critical advance for future structure-based design of non-covalent peptidomimetic inhibitors. Such inhibitors would represent an entirely new antibacterial class that work by switching off the DSB virulence assembly machinery. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A comprehensive study of Superfund program benefits in the Denver and Tampa Bay metropolitan areas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Held, K.; Casper, B.; Siddhanti, S.K.

1995-12-31

The purpose of the study is to evaluate the benefits of the Superfund program in selected geographic areas. The study demonstrates how the cleanup of Superfund sites has improved the overall quality of life of those in the affected communities. The study presents findings on the benefits of Superfund cleanup activity in the Denver, Colorado and Tampa Bay, Florida metropolitan areas. Denver and Tampa Bay were chosen from several areas that the EPA evaluated and screened during the initial phase of the study. These locations were chosen because of a substantial presence of Superfund activities, making it possible to assessmore » the efficacy of the program. Several features make this study unique in terms of its overall goal. The study examines a broad range of benefit categories related to human health, environmental, and socioeconomic effects of Superfund cleanup activities. The study is also designed to assess benefits due to completed, current, and future planned activity at Superfund sites. This assessment covers Federal remedial activities at National Priorities List (NPL) sites, as well as relevant Federal removal actions in the study areas. These benefits are investigated from an area-wide perspective, as opposed to site-by-site, to determine Superfund`s overall effect on the communities in each area. The study consists of two major phases: Phase 1: Screening and ranking 16 prospective geographic areas and selecting Denver and Tampa Bay as the most appropriate areas for in-depth analysis; and Phase 2: Developing methodologies for assessing benefits, collecting relevant data, and analyzing the benefits from Superfund cleanup activity.« less

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas

Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 thatmore » forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.« less

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: Construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile

DOE Office of Scientific and Technical Information (OSTI.GOV)

Street, I.P.; Poulter, C.D.

1990-08-14

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase (IPP isomerase) is an enzyme in isoprene metabolism which catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks for the pathway. The gene encoding IPP isomerase has recently been isolated from Saccharomyces cerevisiae. A heterologous expression system was constructed for the gene and used to overexpress IPP isomerase in Escherichia coli. In transformants carrying the expression vector, IPP isomerase activity was increased by over 100,000-fold relative to that of the untransformed host strain. The overexpressed enzyme constitutes 30-35% of the total soluble cell protein and can be purified to homogeneity in two steps. Recombinantmore » IPP isomerase was indistinguishable from that purified from yeast. 3-(Fluoromethyl)-3-butenyl diphosphate (FIPP) is a specific active-site-directed inhibitor of IPP isomerase from Claviceps purpurea. Inactivation of yeast IPP isomerase by FIPP was active-site-directed, and inhibition resulted in formation of a stoichiometric enzyme-inhibitor complex. The site of covalent attachment in the enzyme-inhibitor complex was determined by inactivating IPP isomerase with (4-{sup 3}H)FIPP, followed by digestion of the labeled enzyme with trypsin and purification of the resulting radioactive peptides by reversed-phase high-performance liquid chromatography. The primary site of attachment was Cys-139.« less

Site Characterization for a Deep Borehole Field Test

NASA Astrophysics Data System (ADS)

Kuhlman, K. L.; Hardin, E. L.; Freeze, G. A.; Sassani, D.; Brady, P. V.

2015-12-01

The US Department of Energy Office of Nuclear Energy is at the beginning of 5-year Deep Borehole Field Test (DBFT) to investigate the feasibility of constructing and characterizing two boreholes in crystalline basement rock to a depth of 5 km (16,400 ft). The concept of deep borehole disposal for radioactive waste has some advantages over mined repositories, including incremental construction and loading, the enhanced natural barriers provided by deep continental crystalline basement, and reduced site characterization. Site characterization efforts need to determine an eligible site that does not have the following disqualifying characteristics: greater than 2 km to crystalline basement, upward vertical fluid potential gradients, presence of economically exploitable natural resources, presence of high permeability connection to the shallow subsurface, and significant probability of future seismic or volcanic activity. Site characterization activities for the DBFT will include geomechanical (i.e., rock in situ stress state, and fluid pressure), geological (i.e., rock and fracture infill lithology), hydrological (i.e., quantity of fluid, fluid convection properties, and solute transport mechanisms), and geochemical (i.e., rock-water interaction and natural tracers) aspects. Both direct (i.e., sampling and in situ testing) and indirect (i.e., borehole geophysical) methods are planned for efficient and effective characterization of these site aspects and physical processes. Borehole-based characterization will be used to determine the variability of system state (i.e., stress, pressure, temperature, and chemistry) with depth, and interpretation of material and system parameters relevant to numerical site simulation. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

Adverse health effects in Canada geese (Branta canadensis) associated with waste from zinc and lead mines in the Tri-State Mining District (Kansas, Oklahoma, and Missouri, USA).

PubMed

van der Merwe, Deon; Carpenter, James W; Nietfeld, Jerome C; Miesner, John F

2011-07-01

Lead and zinc poisoning have been recorded in a variety of bird species, including migrating waterfowl such as Canada Geese (Branta canadensis), at sites contaminated with mine waste from lead and zinc mines in the Tri-State Mining District, Kansas, Oklahoma, and Missouri, USA. The adverse health impacts from mine waste on these birds may, however, be more extensive than is apparent from incidental reports of clinical disease. To characterize health impacts from mine waste on Canada Geese that do not have observable signs of poisoning, four to eight apparently healthy birds per site were collected from four contaminated sites and an uncontaminated reference site, and examined for physical and physiologic evidence of metals poisoning. Tissue concentrations of silver, aluminum, arsenic, barium, cadmium, cobalt, chromium, copper, iron, magnesium, manganese, molybdenum, nickel, lead, selenium, thallium, vanadium, and zinc were determined by inductively coupled plasma mass spectroscopy. Adverse health effects due to lead were characterized by assessing blood δ-aminolevulinic acid dehydratase (ALAD) enzyme activity. Adverse effects associated with zinc poisoning were determined from histologic examination of pancreas tissues. Elevated tissue lead concentrations and inhibited blood ALAD enzyme activities were consistently found in birds at all contaminated sites. Histopathologic signs of zinc poisoning, including fibrosis and vacuolization, were associated with elevated pancreatic zinc concentrations at one of the study sites. Adverse health effects associated with other analyzed elements, or tissue concentrations indicating potentially toxic exposure levels to these elements, were not observed.

The ability of multi-site, multi-depth sacral lateral branch blocks to anesthetize the sacroiliac joint complex.

PubMed

Dreyfuss, Paul; Henning, Troy; Malladi, Niriksha; Goldstein, Barry; Bogduk, Nikolai

2009-01-01

To determine the physiologic effectiveness of multi-site, multi-depth sacral lateral branch injections. Double-blind, randomized, placebo-controlled study. Outpatient pain management center. Twenty asymptomatic volunteers. The dorsal innervation to the sacroiliac joint (SIJ) is from the L5 dorsal ramus and the S1-3 lateral branches. Multi-site, multi-depth lateral branch blocks were developed to compensate for the complex regional anatomy that limited the effectiveness of single-site, single-depth lateral branch injections. Bilateral multi-site, multi-depth lateral branch green dye injections and subsequent dissection on two cadavers revealed a 91% accuracy with this technique. Session 1: 20 asymptomatic subjects had a 25-g spinal needle probe their interosseous (IO) and dorsal sacroiliac (DSI) ligaments. The inferior dorsal SIJ was entered and capsular distension with contrast medium was performed. Discomfort had to occur with each provocation maneuver and a contained arthrogram was necessary to continue in the study. Session 2: 1 week later; computer randomized, double-blind multi-site, multi-depth lateral branch blocks injections were performed. Ten subjects received active (bupivicaine 0.75%) and 10 subjects received sham (normal saline) multi-site, multi-depth lateral branch injections. Thirty minutes later, provocation testing was repeated with identical methodology used in session 1. Presence or absence of pain for ligamentous probing and SIJ capsular distension. Seventy percent of the active group had an insensate IO and DSI ligaments, and inferior dorsal SIJ vs 0-10% of the sham group. Twenty percent of the active vs 10% of the sham group did not feel repeat capsular distension. Six of seven subjects (86%) retained the ability to feel repeat capsular distension despite an insensate dorsal SIJ complex. Multi-site, multi-depth lateral branch blocks are physiologically effective at a rate of 70%. Multi-site, multi-depth lateral branch blocks do not effectively block the intra-articular portion of the SIJ. There is physiological evidence that the intra-articular portion of the SIJ is innervated from both ventral and dorsal sources. Comparative multi-site, multi-depth lateral branch blocks should be considered a potentially valuable tool to diagnose extra-articular SIJ pain and determine if lateral branch radiofrequency neurotomy may assist one with SIJ pain.

Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation.

PubMed

Kumar, Raj; Calhoun, William J

2008-12-01

Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR) is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade. Taken together, site-specific phosphorylation and related kinase pathways play an important role in the action of the GR, and more precise mechanistic information will lead to fuller understanding of the complex nature of gene regulation by the GR- and related transcription factors. This review provides currently available information regarding the role of GR phosphorylation in its action, and highlights the possible underlying mechanisms of action.

How registered nurses, licensed practical nurses and resident aides spend time in nursing homes: An observational study.

PubMed

McCloskey, Rose; Donovan, Cindy; Stewart, Connie; Donovan, Alicia

2015-09-01

Calls for improved conditions in nursing homes have pointed to the importance of optimizing the levels and skills of care providers. Understanding the work of care providers will help to determine if staff are being used to their full potential and if opportunities exist for improved efficiencies. To explore the activities of care providers in different nursing homes and to identify if variations exist within and across homes and shifts. A multi-centre cross-sectional observational work flow study was conducted in seven different nursing homes sites in one Canadian province. Data were collected by a research assistant who conducted 368 h of observation. The research assistant collected data by following an identical route in each site and recording observations on staff activities. Findings indicate staff activities vary across roles, sites and shifts. Licensed practical nurses (nursing assistants) have the greatest variation in their role while registered nurses have the least amount of variability. In some sites both registered nurses and licensed practical nurses perform activities that may be safely delegated to others. Care providers spend as much as 53.7% of their time engaged in non-value added activities. There may be opportunities for registered nurses and licensed practical nurses to delegate some of their activities to non-regulated workers. The time care providers spend in non-value activities suggest there may be opportunities to improve efficiencies within the nursing home setting. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme.

PubMed

Wakamatsu, Taisuke; Sakuraba, Haruhiko; Kitamura, Megumi; Hakumai, Yuichi; Fukui, Kenji; Ohnishi, Kouhei; Ashiuchi, Makoto; Ohshima, Toshihisa

2017-01-15

l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P) + -dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD + Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD + /NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the β-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme. In this study, we determined the three-dimensional structure of l-Trp dehydrogenase, analyzed its various site-directed substitution mutants at residues located in the active site, and obtained the following informative results. Several residues in the active site form a hydrophobic cluster, which may be a part of the hydrophobic core essential for protein folding. To our knowledge, there is no previous report demonstrating that a hydrophobic cluster in the active site of any l-amino acid dehydrogenase may have a critical impact on protein folding. Furthermore, our results suggest that this hydrophobic cluster could strictly accommodate l-Trp. These studies show the structural characteristics of l-Trp dehydrogenase and hence would facilitate novel applications of l-Trp dehydrogenase. Copyright © 2016 American Society for Microbiology.

Stream Phosphorus Dynamics Along a Suburbanizing Gradient in Southern Ontario, Canada

NASA Astrophysics Data System (ADS)

Duval, T. P.

2017-12-01

While it is well known that urban streams are subject to impaired water quality relative to natural analogues, far less research has been directed at stream water quality during the process of (sub-) urbanization. This study determines the role of housing construction activities in Brampton, Canada on the concentration and flux of phosphorus (P) of a headwater stream. Prior to development the stream was engineered with a riffle-pool sequence, riparian plantings, and a floodplain corridor that was lined with sediment fencing. Stream sites were sampled daily over a period of six months at locations representing varying stages of subdivision completion (upper site -active construction; middle site -finished construction and natural vegetation; lower site -finished construction and active construction). A nearby urban stream site developed ten years prior to this study was selected as a reference site. There were no differences in total phosphorus (TP) levels or flux between the suburbanizing and urban streams; however, the forms of P differed between sites. The urban stream TP load was dominated by particulate phosphorus (PP) while suburbanizing stream P was mainly in the dissolved organic phosphorus (DOP) form. The importance of DOP to TP flux increased with the onset of the growing season. TP levels in all stream segments frequently exceeded provincial water quality guidelines during storm events but were generally low during baseflow conditions. During storm events PP and total suspended solid levels in the suburbanizing stream reached levels of the urban stream due to sediment fence failure at several locations along the construction-hillslope interface. Along the suburbanizing gradient, the hydrological connection to a mid-reach zone of no-construction activity / fallow field and native forest resulted in significantly lower P levels than the upper suburbanizing stream site. This suggests that stream channel design features as well as timing of construction activities and the hydrological connection between the stream and construction projects all contribute to downstream export of nutrients and ultimately stream water quality.

Eurasian beaver activity increases water storage, attenuates flow and mitigates diffuse pollution from intensively-managed grasslands.

PubMed

Puttock, Alan; Graham, Hugh A; Cunliffe, Andrew M; Elliott, Mark; Brazier, Richard E

2017-01-15

Beavers are the archetypal keystone species, which can profoundly alter ecosystem structure and function through their ecosystem engineering activity, most notably the building of dams. This can have a major impact upon water resource management, flow regimes and water quality. Previous research has predominantly focused on the activities of North American beaver (Castor canadensis) located in very different environments, to the intensive lowland agricultural landscapes of the United Kingdom and elsewhere in Europe. Two Eurasian beavers (Castor fiber) were introduced to a wooded site, situated on a first order tributary, draining from intensively managed grassland. The site was monitored to understand impacts upon water storage, flow regimes and water quality. Results indicated that beaver activity, primarily via the creation of 13 dams, has increased water storage within the site (holding ca. 1000m 3 in beaver ponds) and beavers were likely to have had a significant flow attenuation impact, as determined from peak discharges (mean 30±19% reduction), total discharges (mean 34±9% reduction) and peak rainfall to peak discharge lag times (mean 29±21% increase) during storm events. Event monitoring of water entering and leaving the site showed lower concentrations of suspended sediment, nitrogen and phosphate leaving the site (e.g. for suspended sediment; average entering site: 112±72mgl -1 , average leaving site: 39±37mgl -1 ). Combined with attenuated flows, this resulted in lower diffuse pollutant loads in water downstream. Conversely, dissolved organic carbon concentrations and loads downstream were higher. These observed changes are argued to be directly attributable to beaver activity at the site which has created a diverse wetland environment, reducing downstream hydrological connectivity. Results have important implications for beaver reintroduction programs which may provide nature based solutions to the catchment-scale water resource management issues that are faced in agricultural landscapes. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

A novel Death Defying Domain in Met entraps the active site of Caspase-3 and blocks apoptosis in hepatocytes

PubMed Central

Ma, Jihong; Zou, Chunbin; Guo, Lida; Seneviratne, Danushka S.; Tan, Xinping; Kwon, Yong-Kook; An, Jiyan; Bowser, Robert; DeFrances, Marie C.; Zarnegar, Reza

2013-01-01

Met, the transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF) is known to function as a potent anti-apoptotic mediator in normal and neoplastic cells. Herein we report that intracellular cytoplasmic tail of Met has evolved to harbor a tandem pair of Caspase-3 cleavage sites, which bait, trap and disable the active site of Caspase-3, thereby blocking the execution of apoptosis. We call this Caspase-3 cleavage motif the ‘Death Defying Domain’ (DDD). This site consists of the following sequence: DNAD-DEVD-T (where the hyphens denote caspase cleavage sites). Through functional and mechanistic studies, we show that upon DDD cleavage by Caspase-3, the resulting DEVD-T peptide acts as a competitive inhibitor and entraps the active site of Caspase-3 akin to DEVD-CHO, which is a potent, synthetic inhibitor of Caspase-3 activity. By gain and loss-of-function studies using restoration of DDD expression in DDD deficient hepatocytic cells, we found that both Caspase-3 sites in DDD are necessary for inhibition of Caspase-3 and promotion of cell survival. Employing mutagenesis studies, we show that DDD could operate independently of Met’s enzymatic activity as determined by using kinase-dead human Met mutant constructs. Studies of both human liver cancer tissues and cell lines uncovered that DDD cleavage and entrapment of Caspase-3 by DDD occur in vivo, further proving that this site has physiological and pathophysiological relevance. Conclusion Our findings show that Met can directly inhibit Caspase-3 via a novel mechanism and promote hepato-cyte survival. Results presented here will further our understanding of the mechanisms that control not only normal tissue homeostasis but also abnormal tissue growth such as cancer and degenerative diseases in which apoptotic caspases are at play. PMID:24122846

Crystallographic Analysis Reveals a Novel Second Binding Site for Trimethoprim in Active Site Double Mutants of Human Dihydrofolate Reductase†,‡

PubMed Central

Cody, Vivian; Pace, Jim; Piraino, Jennifer; Queener, Sherry F.

2011-01-01

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h)DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (Ki 49 nM) compared to hDHFR (Ki 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show Ki values of 593 and 617 nM, respectively; these Ki values are well above both the Ki for pjDHFR and are similar to pcDHFR (Q35K/N64F) and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes. PMID:21684339

Single Atomic Iron Catalysts for Oxygen Reduction in Acidic Media: Particle Size Control and Thermal Activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Hanguang; Hwang, Sooyeon; Wang, Maoyu

To significantly reduce the cost of proton exchange membrane (PEM) fuel cells, current Pt must be replaced by platinum-metal-group (PGM)-free catalysts for the oxygen reduction reaction (ORR) in acid. We report here a new class of high-performance atomic iron dispersed carbon catalysts through controlled chemical doping of iron ions into zinc-zeolitic imidazolate framework (ZIF), a type of metal-organic framework (MOF). The novel synthetic chemistry enables accurate size control of Fe-doped ZIF catalyst particles with a wide range from 20 to 1000 nm without changing chemical properties, which provides a great opportunity to increase the density of active sites that ismore » determined by the particle size. We elucidated the active site formation mechanism by correlating the chemical and structural changes with thermal activation process for the conversion from Fe-N4 complex containing hydrocarbon networks in ZIF to highly active FeNx sites embedded into carbon. A temperature of 800oC was identified as the critical point to start forming pyridinic nitrogen doping at the edge of the graphitized carbon planes. Further increasing heating temperature to 1100oC leads to increase of graphitic nitrogen, generating possible synergistic effect with FeNx sites to promote ORR activity. The best performing catalyst, which has well-defined particle size around 50 nm and abundance of atomic FeNx sites embedded into carbon structures, achieve a new performance milestone for the ORR in acid including a half-wave potential of 0.85 V vs RHE and only 20 mV loss after 10,000 cycles in O2 saturated H2SO4 electrolyte. The new class PGM-free catalyst with approaching activity to Pt holds great promise for future PEM fuel cells.« less

Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Dell, William B.; Agarwal, Pratul K.; Meilleur, Flora

Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. Here, we determined the high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed “pre-bound” molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygenmore » activation. Our results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme–substrate complex.« less

Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase

DOE PAGES

O'Dell, William B.; Agarwal, Pratul K.; Meilleur, Flora

2016-12-22

Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. Here, we determined the high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed “pre-bound” molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygenmore » activation. Our results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme–substrate complex.« less

The instrumental method of plutonium determination

NASA Astrophysics Data System (ADS)

Knyazev, B. B.; Kazachevskiy, I. V.; Solodukhin, V. P.; Lukashenko, S. N.; Knatova, M. K.; Kashirskiy, V. V.

2003-01-01

A method of direct instrumental determination of plutonium isotopes in soil samples is described. For the method a special program of spectra processing and activity calculation had to be prepared. The detection limit of 239+240Pu in absence of interfering radiation is about 200 Bq/kg (by 3.3σ criteria). Examples are given of the method application for the study of radionuclide soil composition in separate objects of Semipalatinsk Nuclear Test Site (SNTS). It is shown that for different objects under study the correlation degree between plutonium and americium activities may change rather substantially.

Granulocyte elastase activity and PGE2 levels in gingival crevicular fluid in relation to the presence of subgingival periodontopathogens in subjects with untreated adult periodontitis.

PubMed

Jin, L J; Söder, P O; Leung, W K; Corbet, E F; Samaranayake, L P; Söder, B; Davies, W I

1999-08-01

This study aimed to determine the association between the levels of granulocyte elastase and prostaglandin E2 (PGE2) in GCE and the concomitant presence of periodontopathogens in untreated adult periodontitis (AP). GCF and subgingival plaque were sampled by paper strips and paper points respectively, from various periodontal sites in 16 AP subjects. Granulocyte elastase activity in GCF was analyzed with a low molecular weight substrate specific for granulocyte elastase, pGluProVal-pNA, and the maximal rate of elastase activity (MR-EA, mAbs/min/site) was calculated. PGE2 levels in GCF were determined by radioimmunoassay. 5 species-specific DNA probes were used to detect the presence of A. actinomyceterncomitans (A.a., ATCC 43718), B. forsythus (B.f, ATCC 43037), P. gingivalis (P.g., ATCC 33277), P. intermedia (P.i., ATCC 33563), and T. denticola (T.d., ATCC 35405), with a sensitivity of 10(3) cells/paper point. No A.a. was detectable from all sites sampled. The predominant combination of species detected was B.f., P.g., P.i. & T.d. and it was significantly higher at periodontitis sites (68%) than at healthy (7%) or gingivitis sites (29%) (p<0.05). Overall, MR-EA values were strongly correlated with PGE2 levels (r=0.655, p<0.001), especially at these periodontitis sites co-infected by B.f., P.g., P.i. & T.d. (r=0.722, p<0.001). The periodontitis sites co-infected by the 4 species were observable from 15 subjects. These sites were sub-grouped into 8 subjects with a high MR-EA and 7 subjects with a low MR-EA. The PGE2 levels in the high MR-EA group were significantly higher than in the low MR-EA group (p<0.05). No significant differences in clinical or bacterial data were found between the two groups. While within the high MR-EA group, similar results were found between the paired periodontitis sites in each subject with highest and lowest MR-EA values. This study shows that the local host response to bacterial challenge in untreated periodontal pockets is diverse in terms of the intensity of inflammatory response measured by granulocyte elastase and PGE2 levels in GCE A more thorough evaluation of the risk for active periodontal disease may involve the combined approaches to the test of the dynamic bacteria-host relations.

Coupling of Realistic Rate Estimates with Genomics for Assessing Contaminant Attenuation and Long-Term Plume Containment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colwell, F.S.; Crawford, R.L.; Sorenson, K.

2005-09-01

Acceptance of monitored natural attenuation (MNA) as a preferred treatment technology saves significant site restoration costs for DOE. However, in order to be accepted MNA requires direct evidence of which processes are responsible for the contaminant loss and also the rates of the contaminant loss. Our proposal aims to: 1) provide evidence for one example of MNA, namely the disappearance of the dissolved trichloroethylene (TCE) from the Snake River Plain aquifer (SRPA) at the Idaho National Laboratory’s Test Area North (TAN) site, 2) determine the rates at which aquifer microbes can co-metabolize TCE, and 3) determine whether there are othermore » examples of natural attenuation of chlorinated solvents occurring at DOE sites. To this end, our research has several objectives. First, we have conducted studies to characterize the microbial processes that are likely responsible for the co-metabolic destruction of TCE in the aquifer at TAN (University of Idaho and INL). Second, we are investigating realistic rates of TCE co-metabolism at the low catabolic activities typical of microorganisms existing under aquifer conditions (INL). Using the co-metabolism rate parameters derived in low-growth bioreactors, we will complete the models that predict the time until background levels of TCE are attained in the aquifer at TAN and validate the long-term stewardship of this plume. Coupled with the research on low catabolic activities of co-metabolic microbes we are determining the patterns of functional gene expression by these cells, patterns that may be used to diagnose the co-metabolic activity in the SRPA or other aquifers.« less

High Throughput Determination of Ricinine Abrine and Alpha ...

EPA Pesticide Factsheets

Analytical Method This document provides the standard operating procedure for determination of ricinine (RIC), abrine (ABR), and α-amanitin (AMAN) in drinking water by isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS). This method is designed to support site-specific cleanup goals of environmental remediation activities following a homeland security incident involving one or a combination of these analytes.

A measurement of the magnetic field direction at the site of major flares

NASA Technical Reports Server (NTRS)

Lundstedt, H.

1982-01-01

Lundstedt et al. (1981) showed that the direction of the photospheric magnetic field at the site of a flare is a good predictor of the solar wind velocity observed at earth four days later. It is described here how the field direction was obtained, and possible errors involved in the determination of the angle are discussed. The discussion also includes a characterization of the solar active regions.

Satellite Power System (SPS) laser studies. Volume 2: Meteorological effects on laser beam propagation and direct solar pumped lasers for the SPS

NASA Technical Reports Server (NTRS)

Beverly, R. E., III

1980-01-01

The primary emphasis of this research activity was to investigate the effect of the environment on laser power transmission/reception from space to ground. Potential mitigation techniques to minimize the environment effect by a judicious choice of laser operating parameters was investigated. Using these techniques, the availability of power at selected sites was determined using statistical meteorological data for each site.

Determinants of community structure of zooplankton in heavily polluted river ecosystems

NASA Astrophysics Data System (ADS)

Xiong, Wei; Li, Jie; Chen, Yiyong; Shan, Baoqing; Wang, Weimin; Zhan, Aibin

2016-02-01

River ecosystems are among the most affected habitats globally by human activities, such as the release of chemical pollutants. However, it remains largely unknown how and to what extent many communities such as zooplankton are affected by these environmental stressors in river ecosystems. Here, we aim to determine major factors responsible for shaping community structure of zooplankton in heavily polluted river ecosystems. Specially, we use rotifers in the Haihe River Basin (HRB) in North China as a case study to test the hypothesis that species sorting (i.e. species are “filtered” by environmental factors and occur at environmental suitable sites) plays a key role in determining community structure at the basin level. Based on an analysis of 94 sites across the plain region of HRB, we found evidence that both local and regional factors could affect rotifer community structure. Interestingly, further analyses indicated that local factors played a more important role in determining community structure. Thus, our results support the species sorting hypothesis in highly polluted rivers, suggesting that local environmental constraints, such as environmental pollution caused by human activities, can be stronger than dispersal limitation caused by regional factors to shape local community structure of zooplankton at the basin level.

Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.

PubMed Central

Aghajari, N.; Feller, G.; Gerday, C.; Haser, R.

1998-01-01

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase. PMID:9541387

Increased oxidative stress in human fetal membranes overlying the cervix from term non-labouring and post labour deliveries.

PubMed

Chai, M; Barker, G; Menon, R; Lappas, M

2012-08-01

Enzymatic breakdown of the collagen-rich extracellular matrix (ECM) that connects the amnion and chorion layers of the fetal membranes is one of the key events leading to rupture of membranes. Oxidant stress caused by increased formation of reactive oxygen species and/or reduced antioxidant capacity may predispose to membrane rupture, a major cause of preterm birth. The aim of this study was to determine the effect of human labour and supracervical (SC) apposition on antioxidant enzymes and 8-isoprostane (a marker of lipid peroxidation). To determine the effect of human labour on oxidative stress status, fetal membranes from the SC site (SCS) were collected from women at term Caesarean section (no labour), and from the site of membrane rupture (SOR) after spontaneous labour onset and delivery (post labour). To determine the effect of SC apposition on oxidative stress status, amnion was collected from the SCS and a distal site (DS) in women at term Caesarean section in the absence of labour. The release of 8-isoprostane was significantly higher in amnion from the SCS compared to DS, and in fetal membranes from the SOR compared to the SCS. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity were lower in amnion from the SC compared to DS. SOD gene expression and enzyme activity were lower in fetal membranes after labour. There was no difference in expression or activity in catalase, GPx and glutathione reductase (GSR) between no labour and post labour fetal membranes. In primary amnion cells, SOD supplementation significantly augmented IL-1β induced MMP-9 expression and activity. In summary, non-labouring SC fetal membranes are characterised by reduced antioxidant enzyme activity when compared to distal membranes, and, as such, may be more susceptible to oxidative damage and thus membrane rupture. Copyright © 2012 Elsevier Ltd. All rights reserved.

Regulation of MVM NS1 by protein kinase C: impact of mutagenesis at consensus phosphorylation sites on replicative functions and cytopathic effects.

PubMed

Corbau, R; Duverger, V; Rommelaere, J; Nüesch, J P

2000-12-05

Minute virus of mice NS1, an 83-kDa mainly nuclear phosphoprotein, is the only viral nonstructural protein required in all cell types and it is involved in multiple processes necessary for virus propagation. The diversity of functions assigned to NS1, together with the variation of its complex phosphorylation pattern during infection, suggested that the various activities of NS1 could be regulated by distinct phosphorylation events. So far, it has been demonstrated that NS1 replicative functions, in particular, DNA-unwinding activities, are regulated by protein kinase C (PKC), as exemplified by the modulation of NS1 helicase activity by PKClambda phosphorylation. In order to determine further impact of phosphorylation on NS1 functions, including the induction of cytopathic effects, a mutational approach was pursued in order to produce NS1 variants harboring amino acid substitutions at candidate PKC target residues. Besides the determination of two additional in vivo phosphorylation sites in NS1, this mutagenesis allowed the segregation of distinct NS1 functions from one another, generating NS1 variants with a distinct activity profile. Thus, we obtained NS1 mutants that were fully proficient for trans activation of the viral P38 promoter, while being impaired in their replicative functions. Moreover, the alterations of specific PKC phosphorylation sites gave rise to NS1 polypeptides that exerted reduced cytotoxicity, leading to sustained gene expression, while keeping functions necessary for progeny virus production, i.e., viral DNA replication and activation of the capsid gene promoter. These data suggested that in the course of a viral infection, NS1 may undergo a shift from productive to cytotoxic functions as a result of a phosphorylation-dependent regulation. Copyright 2000 Academic Press.

Regulated expression of a repressor protein: FadR activates iclR.

PubMed Central

Gui, L; Sunnarborg, A; LaPorte, D C

1996-01-01

The control of the glyoxylate bypass operon (aceBAK) of Escherichia coli is mediated by two regulatory proteins, IclMR and FadR. IclMR is a repressor protein which has previously been shown to bind to a site which overlaps the aceBAK promoter. FAR is a repressor/activator protein which participates in control of the genes of fatty acid metabolism. A sequence just upstream of the iclR promoter bears a striking resemblance to FadR binding sites found in the fatty acid metabolic genes. The in vitro binding specificity of FadR, determined by oligonucleotide selection, was in good agreement with the sequences of these sites. The ability of FadR to bind to the site associated with iclR was demonstrated by gel shift and DNase I footprint analyses. Disruption of FadR or inactivation of the FadR binding site of iclR decreased the expression of an iclR::lacZ operon fusion, indicating that FadR activates the expression of iclR. It has been reported that disruption of fadR increases the expression of aceBAK. We observed a similar increase when we inactivated the FadR binding site of an iclR+ allele. This result suggests that FadR regulates aceBAK indirectly by altering the expression of IclR. PMID:8755903

A Methodology to Evaluate Ecological Resources and Risk Using Two Case Studies at the Department of Energy's Hanford Site

NASA Astrophysics Data System (ADS)

Burger, Joanna; Gochfeld, Michael; Bunn, Amoret; Downs, Janelle; Jeitner, Christian; Pittfield, Taryn; Salisbury, Jennifer; Kosson, David

2017-03-01

An assessment of the potential risks to ecological resources from remediation activities or other perturbations should involve a quantitative evaluation of resources on the remediation site and in the surrounding environment. We developed a risk methodology to rapidly evaluate potential impact on ecological resources for the U.S. Department of Energy's Hanford Site in southcentral Washington State. We describe the application of the risk evaluation for two case studies to illustrate its applicability. The ecological assessment involves examining previous sources of information for the site, defining different resource levels from 0 to 5. We also developed a risk rating scale from non-discernable to very high. Field assessment is the critical step to determine resource levels or to determine if current conditions are the same as previously evaluated. We provide a rapid assessment method for current ecological conditions that can be compared to previous site-specific data, or that can be used to assess resource value on other sites where ecological information is not generally available. The method is applicable to other Department of Energy's sites, where its development may involve a range of state regulators, resource trustees, Tribes and other stakeholders. Achieving consistency across Department of Energy's sites for valuation of ecological resources on remediation sites will assure Congress and the public that funds and personnel are being deployed appropriately.

A Methodology to Evaluate Ecological Resources and Risk Using Two Case Studies at the Department of Energy's Hanford Site.

PubMed

Burger, Joanna; Gochfeld, Michael; Bunn, Amoret; Downs, Janelle; Jeitner, Christian; Pittfield, Taryn; Salisbury, Jennifer; Kosson, David

2017-03-01

An assessment of the potential risks to ecological resources from remediation activities or other perturbations should involve a quantitative evaluation of resources on the remediation site and in the surrounding environment. We developed a risk methodology to rapidly evaluate potential impact on ecological resources for the U.S. Department of Energy's Hanford Site in southcentral Washington State. We describe the application of the risk evaluation for two case studies to illustrate its applicability. The ecological assessment involves examining previous sources of information for the site, defining different resource levels from 0 to 5. We also developed a risk rating scale from non-discernable to very high. Field assessment is the critical step to determine resource levels or to determine if current conditions are the same as previously evaluated. We provide a rapid assessment method for current ecological conditions that can be compared to previous site-specific data, or that can be used to assess resource value on other sites where ecological information is not generally available. The method is applicable to other Department of Energy's sites, where its development may involve a range of state regulators, resource trustees, Tribes and other stakeholders. Achieving consistency across Department of Energy's sites for valuation of ecological resources on remediation sites will assure Congress and the public that funds and personnel are being deployed appropriately.

Source identification of uranium-containing materials at mine legacy sites in Portugal.

PubMed

Keatley, A C; Martin, P G; Hallam, K R; Payton, O D; Awbery, R; Carvalho, F P; Oliveira, J M; Silva, L; Malta, M; Scott, T B

2018-03-01

Whilst prior nuclear forensic studies have focused on identifying signatures to distinguish between different uranium deposit types, this paper focuses on providing a scientific basis for source identification of materials from different uranium mine sites within a single region, which can then be potentially used within nuclear forensics. A number of different tools, including gamma spectrometry, alpha spectrometry, mineralogy and major and minor elemental analysis, have been utilised to determine the provenance of uranium mineral samples collected at eight mine sites, located within three different uranium provinces, in Portugal. A radiation survey was initially conducted by foot and/or unmanned aerial vehicle at each site to assist sample collection. The results from each mine site were then compared to determine if individual mine sites could be distinguished based on characteristic elemental and isotopic signatures. Gamma and alpha spectrometry were used to differentiate between samples from different sites and also give an indication of past milling and mining activities. Ore samples from the different mine sites were found to be very similar in terms of gangue and uranium mineralogy. However, rarer minerals or specific impurity elements, such as calcium and copper, did permit some separation of the sites examined. In addition, classification rates using linear discriminant analysis were comparable to those in the literature. Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.

Structure determination of uracil-DNA N-glycosylase from Deinococcus radiodurans in complex with DNA.

PubMed

Pedersen, Hege Lynum; Johnson, Kenneth A; McVey, Colin E; Leiros, Ingar; Moe, Elin

2015-10-01

Uracil-DNA N-glycosylase (UNG) is a DNA-repair enzyme in the base-excision repair (BER) pathway which removes uracil from DNA. Here, the crystal structure of UNG from the extremophilic bacterium Deinococcus radiodurans (DrUNG) in complex with DNA is reported at a resolution of 1.35 Å. Prior to the crystallization experiments, the affinity between DrUNG and different DNA oligonucleotides was tested by electrophoretic mobility shift assays (EMSAs). As a result of this analysis, two 16 nt double-stranded DNAs were chosen for the co-crystallization experiments, one of which (16 nt AU) resulted in well diffracting crystals. The DNA in the co-crystal structure contained an abasic site (substrate product) flipped into the active site of the enzyme, with no uracil in the active-site pocket. Despite the high resolution, it was not possible to fit all of the terminal nucleotides of the DNA complex into electron density owing to disorder caused by a lack of stabilizing interactions. However, the DNA which was in contact with the enzyme, close to the active site, was well ordered and allowed detailed analysis of the enzyme-DNA interaction. The complex revealed that the interaction between DrUNG and DNA is similar to that in the previously determined crystal structure of human UNG (hUNG) in complex with DNA [Slupphaug et al. (1996). Nature (London), 384, 87-92]. Substitutions in a (here defined) variable part of the leucine loop result in a shorter loop (eight residues instead of nine) in DrUNG compared with hUNG; regardless of this, it seems to fulfil its role and generate a stabilizing force with the minor groove upon flipping out of the damaged base into the active site. The structure also provides a rationale for the previously observed high catalytic efficiency of DrUNG caused by high substrate affinity by demonstrating an increased number of long-range electrostatic interactions between the enzyme and the DNA. Interestingly, specific interactions between residues in the N-terminus of a symmetry-related molecule and the complementary DNA strand facing away from the active site were also observed which seem to stabilize the enzyme-DNA complex. However, the significance of this observation remains to be investigated. The results provide new insights into the current knowledge about DNA damage recognition and repair by uracil-DNA glycosylases.

A hinge migration mechanism unlocks the evolution of green-to-red photoconversion in GFP-like proteins

DOE PAGES

Kim, Hanseong; Zou, Taisong; Modi, Chintan; ...

2014-12-31

In proteins, functional divergence involves mutations that modify structure and dynamics. In this paper, we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. Crystallographic structures were determined for several reconstructed ancestral proteins belonging to a GFP class frequently employed in superresolution microscopy. Their chain flexibility was analyzed using molecular dynamics and perturbation response scanning. The green-to-red photoconvertible phenotype appears to have arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the βmore » barrel fold. The allosterically coupled mutational sites provide active site conformational mobility via epistasis. We propose that light-induced chromophore twisting is enhanced in a reverse-protonated subpopulation, activating internal acid-base chemistry and backbone cleavage to enlarge the chromophore. Finally, dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities.« less

Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases.

PubMed

Kjaergaard, Christian H; Qayyum, Munzarin F; Wong, Shaun D; Xu, Feng; Hemsworth, Glyn R; Walton, Daniel J; Young, Nigel A; Davies, Gideon J; Walton, Paul H; Johansen, Katja Salomon; Hodgson, Keith O; Hedman, Britt; Solomon, Edward I

2014-06-17

Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy.

An Exploration of the Use of Branding to Shape Institutional Image in the Marketing Activities of Faith-Based Higher Education Institutions

ERIC Educational Resources Information Center

Tolbert, Dawn

2014-01-01

Modern higher education includes student-consumers who shop for educational opportunities and institutions that actively market themselves. This study examined the marketing of faith-based institutions to determine how faith-related missions are reflected in the printed recruitment materials, Web sites, and admissions portals of the 112 member…

Carbonylation of Mitochondrial Aconitase with 4-Hydroxy-2-(E)-nonenal: Localization and Relative Reactivity of Addition Sites

PubMed Central

Liu, Qingyuan; Simpson, David C.; Gronert, Scott

2013-01-01

Mass spectrometry was used to investigate the effects of exposing mitochondrial aconitase (ACO2) to the membrane lipid peroxidation product, 4-hydroxy-2-(E)-nonenal (HNE). ACO2 was selected for this study because (1) it is known to be inactivated by HNE, (2) elevated concentrations of HNE-adducted ACO2 have been associated with disease states, (3) extensive structural information is available, and (4) the iron-sulfur cluster in ACO2 offers a critical target for HNE adduction. The aim of this study was to relate the inactivation of ACO2 by HNE to structural features. Initially, western blotting and an enzyme activity assay were used to assess aggregate effects and then gel electrophoresis, in-gel digestion, and tandem mass spectrometry were used to identify HNE addition sites. HNE addition reaction rates were determined for the most significant sites using the iTRAQ approach. The most reactive sites were Cys358, Cys421, and Cys424, the three iron-sulfur cluster-coordinating cysteines, Cys99, the closest non-ligated cysteine to the cluster, and Cys565, which is located in the cleft leading to the active site. Interestingly, both enzyme activity assay and iTRAQ relative abundance plots appeared to be trending toward horizontal asymptotes, rather than completion. PMID:23518448

Site Safety Plan for Lawrence Livermore National Laboratory CERCLA investigations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bainer, R.; Duarte, J.

1993-07-01

The safety policy of LLNL is to take every reasonable precaution in the performance of work to protect the environment and the health and safety of employees and the public, and to prevent property damage. With respect to hazardous agents, this protection is provided by limiting human exposures, releases to the environment, and contamination of property to levels that are as low as reasonably achievable (ALARA). It is the intent of this Plan to supply the broad outline for completing environmental investigations within ALARA guidelines. It may not be possible to determine actual working conditions in advance of the work;more » therefore, planning must allow the opportunity to provide a range of protection based upon actual working conditions. Requirements will be the least restrictive possible for a given set of circumstances, such that work can be completed in an efficient and timely fashion. Due to the relatively large size of the LLNL Site and the different types of activities underway, site-specific Operational Safety Procedures (OSPs) will be prepared to supplement activities not covered by this Plan. These site-specific OSPs provide the detailed information for each specific activity and act as an addendum to this Plan, which provides the general plan for LLNL Main Site operation.« less

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Computational Tools for Allosteric Drug Discovery: Site Identification and Focus Library Design.

PubMed

Huang, Wenkang; Nussinov, Ruth; Zhang, Jian

2017-01-01

Allostery is an intrinsic phenomenon of biological macromolecules involving regulation and/or signal transduction induced by a ligand binding to an allosteric site distinct from a molecule's active site. Allosteric drugs are currently receiving increased attention in drug discovery because drugs that target allosteric sites can provide important advantages over the corresponding orthosteric drugs including specific subtype selectivity within receptor families. Consequently, targeting allosteric sites, instead of orthosteric sites, can reduce drug-related side effects and toxicity. On the down side, allosteric drug discovery can be more challenging than traditional orthosteric drug discovery due to difficulties associated with determining the locations of allosteric sites and designing drugs based on these sites and the need for the allosteric effects to propagate through the structure, reach the ligand binding site and elicit a conformational change. In this study, we present computational tools ranging from the identification of potential allosteric sites to the design of "allosteric-like" modulator libraries. These tools may be particularly useful for allosteric drug discovery.

Workplace health and safety regulations: Impact of enforcement and consultation on workers' compensation claims rates in Washington State.

PubMed

Baggs, James; Silverstein, Barbara; Foley, Michael

2003-05-01

There has been considerable debate in the public policy arena about the appropriate mix of regulatory enforcement and consultation in achieving desired health and safety behavior across industries. Recently there has been a shift in federal policy toward voluntary approaches and constraining the scope of enforcement programs, although there is little evidence that this might improve health and safety outcomes. To address this, we examined changes in lost time workers compensation claims rates for Washington State employers who had (1) no OSHA State Plan (WISHA) activity, (2) enforcement, (3) consultation, and (4) both types of visits. Compensable claims rates, hours, and WISHA activity were determined for each employer account with a single business location that had payroll hours reported for every quarter from 1997-2000 and more than 10 employees. We used a generalized estimating equations (GEE) approach to Poisson regression to model the association between WISHA activity and claims rate controlling for other external factors. Controlling for previous claims rate and average size, claims rates for employers with WISHA enforcement activity declined 22.5% in fixed site industry SIC codes compared to 7% among employers with no WISHA activity (P < 0.05), and in non-fixed site SICs (e.g., construction) claims rates declined 12.8% for employers with enforcement activity compared to a 7.4% decline for those with no WISHA activity (P > 0.10). WISHA consultation activity was not associated with a greater decline in compensable claims rates (-2.3% for fixed sites and +3.5% for non-fixed sites). WISHA activity did not adversely affect worksite survivability through the study period. Enforcement inspections are significantly associated with decreasing compensable workers compensation claims rates especially for fixed site employers. We were unable to identify an association between consultation activities and decreasing claims rates. Copyright 2003 Wiley-Liss, Inc.

Tetramethylpyrazine-Inducible Promoter Region from Rhodococcus jostii TMP1.

PubMed

Stanislauskienė, Rūta; Kutanovas, Simonas; Kalinienė, Laura; Bratchikov, Maksim; Meškys, Rolandas

2018-06-25

An inducible promoter region, P TTMP (tetramethylpyrazine [TTMP]), has been identified upstream of the tpdABC operon, which contains the genes required for the initial degradation of 2,3,5,6-tetramethylpyrazine in Rhodococcus jostii TMP1 bacteria. In this work, the promoter region was fused with the gene for the enhanced green fluorescent protein (EGFP) to investigate the activity of P TTMP by measuring the fluorescence of bacteria. The highest promoter activity was observed when bacteria were grown in a nutrient broth (NB) medium supplemented with 5 mM 2,3,5,6-tetramethylpyrazine for 48 h. Using a primer extension reaction, two transcriptional start sites for tpdA were identified, and the putative −35 and −10 promoter motifs were determined. The minimal promoter along with two 15 bp long direct repeats and two 7 bp inverted sequences were identified. Also, the influence of the promoter elements on the activity of P TTMP were determined using site-directed mutagenesis. Furthermore, P TTMP was shown to be induced by pyrazine derivatives containing methyl groups in the 2- and 5-positions of the heterocyclic ring, in the presence of the LuxR family transcriptional activator TpdR.

STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF CIRCULAR PERMUTATION ON THE ACTIVE SITE OF OLD YELLOW ENZYME.

PubMed

Daugherty, Ashley B; Horton, John R; Cheng, Xiaodong; Lutz, Stefan

2015-02-06

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme's catalytic performance. Termini relocation into four regions of the protein (sectors I-IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I-III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location, but also provide a possible explanation for the catalytic gains in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290-310) of OYE1 which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such active site remodeling does not negatively impact the enzyme's activity and stereoselectivity, nor does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereo-selectivity for ( S )-carvone reduction. Our findings demonstrate the contribution of loop β6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.

Structural and Functional Consequences of Circular Permutation on the Active Site of Old Yellow Enzyme

DOE PAGES

Daugherty, Ashley B.; Horton, John R.; Cheng, Xiaodong; ...

2014-12-09

Circular permutation of the NADPH-dependent oxidoreductase Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) can significantly enhance the enzyme’s catalytic performance. Termini relocation into four regions of the protein (sectors I–IV) near the active site has proven effective in altering enzyme function. To better understand the structural consequences and rationalize the observed functional gains in these OYE1 variants, we selected representatives from sectors I–III for further characterization by biophysical methods and X-ray crystallography. These investigations not only show trends in enzyme stability and quaternary structure as a function of termini location but also provide a possible explanation for the catalytic gainsmore » in our top-performing OYE variant (new N-terminus at residue 303; sector III). Crystallographic analysis indicates that termini relocation into sector III affects the loop β6 region (amino acid positions: 290–310) of OYE1, which forms a lid over the active site. Peptide backbone cleavage greatly enhances local flexibility, effectively converting the loop into a tether and consequently increasing the environmental exposure of the active site. Interestingly, such an active site remodeling does not negatively impact the enzyme’s activity and stereoselectivity; neither does it perturb the conformation of other key active site residues with the exception of Y375. These observations were confirmed in truncation experiments, deleting all residues of the loop β6 region in our OYE variant. Intrigued by the finding that circular permutation leaves most of the key catalytic residues unchanged, we also tested OYE permutants for possible additive or synergistic effects of amino acid substitutions. Distinct functional changes in these OYE variants were detected upon mutations at W116, known in native OYE1 to cause inversion of diastereoselectivity for (S)-carvone reduction. In conclusion, our findings demonstrate the contribution of loop β6 toward determining the stereoselectivity of OYE1, an important insight for future OYE engineering efforts.« less

Association between Latest Activated Sites in the Left Ventricle and Akinetic Segments in Patients with Ischemic Cardiomyopathy.

PubMed

Sadeghian, Hakimeh; Kousari, Aliasghar; Majidi, Shahla; Rezvanfard, Mehrnaz; Kazemisaeid, Ali; Moezzi, Seyed Ali; Vasheghani Farahani, Ali; Abdar Esfahani, Morteza; Sahebjam, Mohammad; Zoroufian, Arezoo; Sadeghian, Afsaneh

2016-07-06

Background: It is not clear whether the latest activation sites in the left ventricle (LV) are matched with infracted regions in patients with ischemic cardiomyopathy (ICM). We aimed to investigate whether the latest activation sites in the LV are in agreement with the region of akinesia in patients with ICM. Methods: Data were analyzed in 106 patients (age = 60.5 ± 12.1 y, male = 88.7%) with ICM (ejection fraction ≤ 35%) who were refractory to pharmacological therapy and were referred to the echocardiography department for an evaluation of the feasibility of cardiac resynchronization therapy. Wall motion abnormalities, time to peak systolic myocardial velocity (Ts) of 6 basal and 6 mid-portion segments of the LV, and 4 frequently used dyssynchrony indices were measured using 2-dimensional echocardiography and tissue Doppler imaging (TDI). To evaluate the influence of the electrocardiographic pattern, we categorized the patients into 2 groups: patients with QRS ≤ 120 ms and those with QRS >120 ms. Results: A total of 1 272 segments were studied. The latest activation sites (with longest Ts) were most frequently located in the mid-anterior (n = 32, 30.2%) and basal-anterior segments (n = 29, 27.4%), while the most common sites of akinesia were the mid-anteroseptal (n = 65, 61.3%) and mid-septal (n = 51, 48.1%) segments. Generally, no significant concordance was found between the latest activated segments and akinesia either in all the patients or in the QRS groups. Detailed analysis within the segments indicated a good agreement between akinesia and delayed activation in the basal-lateral segment solely in the patients with QRS duration ≤ 120 ms (Φ = 0.707; p value ≤ 0.001). Conclusion: The akinetic segment on 2-dimensional echocardiogram was not matched with the latest activation sites in the LV determined by TDI in patients with ICM.

Association between Latest Activated Sites in the Left Ventricle and Akinetic Segments in Patients with Ischemic Cardiomyopathy

PubMed Central

Sadeghian, Hakimeh; Kousari, Aliasghar; Majidi, Shahla; Rezvanfard, Mehrnaz; Kazemisaeid, Ali; Moezzi, Seyed Ali; Vasheghani Farahani, Ali; Abdar Esfahani, Morteza; Sahebjam, Mohammad; Zoroufian, Arezoo; Sadeghian, Afsaneh

2016-01-01

Background: It is not clear whether the latest activation sites in the left ventricle (LV) are matched with infracted regions in patients with ischemic cardiomyopathy (ICM). We aimed to investigate whether the latest activation sites in the LV are in agreement with the region of akinesia in patients with ICM. Methods: Data were analyzed in 106 patients (age = 60.5 ± 12.1 y, male = 88.7%) with ICM (ejection fraction ≤ 35%) who were refractory to pharmacological therapy and were referred to the echocardiography department for an evaluation of the feasibility of cardiac resynchronization therapy. Wall motion abnormalities, time to peak systolic myocardial velocity (Ts) of 6 basal and 6 mid-portion segments of the LV, and 4 frequently used dyssynchrony indices were measured using 2-dimensional echocardiography and tissue Doppler imaging (TDI). To evaluate the influence of the electrocardiographic pattern, we categorized the patients into 2 groups: patients with QRS ≤ 120 ms and those with QRS >120 ms. Results: A total of 1 272 segments were studied. The latest activation sites (with longest Ts) were most frequently located in the mid-anterior (n = 32, 30.2%) and basal-anterior segments (n = 29, 27.4%), while the most common sites of akinesia were the mid-anteroseptal (n = 65, 61.3%) and mid-septal (n = 51, 48.1%) segments. Generally, no significant concordance was found between the latest activated segments and akinesia either in all the patients or in the QRS groups. Detailed analysis within the segments indicated a good agreement between akinesia and delayed activation in the basal-lateral segment solely in the patients with QRS duration ≤ 120 ms (Φ = 0.707; p value ≤ 0.001). Conclusion: The akinetic segment on 2-dimensional echocardiogram was not matched with the latest activation sites in the LV determined by TDI in patients with ICM. PMID:27956911

Structural Basis of the Induced-Fit Mechanism of 1,4-Dihydroxy-2-Naphthoyl Coenzyme A Synthase from the Crotonase Fold Superfamily

PubMed Central

Li, Jie; Li, Yan; Jiang, Ming; Zhou, Jiahai; Guo, Zhihong

2013-01-01

1, 4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase fold enzyme with an implicated role of conformational changes in catalysis. We have identified these conformational changes by determining the structures of its Escherichia coli and Synechocystis sp. PCC6803 orthologues in complex with a product analog. The structural changes include the folding of an active-site loop into a β-hairpin and significant reorientation of a helix at the carboxy terminus. Interestingly, a new interface is formed between the ordered loop and the reoriented helix, both of which also form additional interactions with the coenzyme A moiety of the ligand. Site-directed mutation of the amino acid residues involved in these ligand-induced interactions significantly diminishes the enzyme activity. These results suggest a catalytically essential induced-fit that is likely initiated by the enzyme-ligand interactions at the active site. PMID:23658663

NRC Waste Incidental to Reprocessing Program: Overview of Consultation and Monitoring Activities at the Idaho National Laboratory and the Savannah River Site - What We Have Learned - 12470

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suber, Gregory

2012-07-01

In 2005 the U.S. Nuclear Regulatory Commission (NRC) began to implement a new set of responsibilities under the Ronald W. Reagan National Defense Authorization Act (NDAA) of Fiscal Year 2005. Section 3116 of the NDAA requires the U.S. Department of Energy (DOE) to consult with the NRC for certain non-high level waste determinations and also requires NRC to monitor DOE's disposal actions related to those determinations. In Fiscal Year 2005, the NRC staff began consulting with DOE and completed reviews of draft waste determinations for salt waste at the Savannah River Site. In 2006, a second review was completed onmore » tank waste residuals including sodium-bearing waste at the Idaho Nuclear Technology and Engineering Center Tank Farm at the Idaho National Laboratory. Monitoring Plans were developed for these activities and the NRC is actively monitoring disposal actions at both sites. NRC is currently in consultation with DOE on the F-Area Tank Farm closure and anticipates entering consultation on the H-Area Tank Farm at the Savannah River Site. This paper presents, from the NRC perspective, an overview of how the consultation and monitoring process has evolved since its conception in 2005. It addresses changes in methods and procedures used to collect and develop information used by the NRC in developing the technical evaluation report and monitoring plan under consultation and the implementation the plan under monitoring. It will address lessons learned and best practices developed throughout the process. The NDAA has presented significant challenges for the NRC and DOE. Past and current successes demonstrate that the NDAA can achieve its intended goal of facilitating tank closure at DOE legacy defense waste sites. The NRC believes many of the challenges in performing the WD reviews have been identified and addressed. Lessons learned have been collected and documented throughout the review process. Future success will be contingent on each agencies commitment to consistently apply the lessons learned and continue to create an open and collaborative work environment to maintain the process of continuous improvement. (authors)« less

Characterization of 5' end of human thromboxane receptor gene. Organizational analysis and mapping of protein kinase C--responsive elements regulating expression in platelets.

PubMed

D'Angelo, D D; Davis, M G; Houser, W A; Eubank, J J; Ritchie, M E; Dorn, G W

1995-09-01

Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest that the mechanism for previously described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcription in platelet-progenitor cells.

Cloning and characterization of a novel acidic cutinase from Sirococcus conigenus.

PubMed

Nyyssölä, Antti; Pihlajaniemi, Ville; Häkkinen, Mari; Kontkanen, Hanna; Saloheimo, Markku; Nakari-Setälä, Tiina

2014-04-01

A cutinase gene (ScCut1) was amplified by PCR from the genomic DNA of the ascomycetous plant pathogen Sirococcous conigenus VTT D-04989 using degenerate primers designed on the basis of conserved segments of known cutinases and cutinase-like enzymes. No introns or N- or O-glycosylation sites could be detected by analysis of the ScCut1 gene sequence. The alignment of ScCut1 with other fungal cutinases indicated that ScCut1 contained the conserved motif G-Y-S-Q-G surrounding the active site serine as well as the aspartic acid and histidine residues of the cutinase active site. The gene was expressed in Pichia pastoris, and the recombinantly produced ScCut1 enzyme was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His-tag translationally fused to the protein. The purified ScCut1 exhibited activity at acidic pH. The K(m) and V(max) values determined for pNP-butyrate esterase activity at pH 4.5 were 1.7 mM and 740 nkat mg⁻¹, respectively. Maximal activities were determined at between pH 4.7 and 5.2 and at between pH 4.1 and 4.6 with pNP-butyrate and tritiated cutin as the substrates, respectively. With both substrates, the enzyme was active over a broad pH range (between pH 3.0 and 7.5). Activity could still be detected at pH 3.0 both with tritiated cutin and with p-nitrophenyl butyrate (relative activity of 25 %) as the substrates. ScCut1 showed activity towards shorter (C2 to C3) fatty acid esters of p-nitrophenol than towards longer ones. Circular dichroism analysis suggested that the denaturation of ScCut1 by heating the protein sample to 80 °C was to a great extent reversible.

Ionic Diffusion in Cu6PS5Br Studied by 63Cu NMR

NASA Astrophysics Data System (ADS)

Ohki, H.; Harazono, K.; Erata, T.; Tasaki, A.; Ikeda, R.

1993-10-01

Applying 63Cu NMR technique, we observed exchange between the nonequivalent copper sites in crystalline Cu6PS5Br, known as a member of the mineral "argyrodite". Below 200 K, where the motion of the copper (I) ion is slow, we could distinguish several nonequivalent copper sites. On increasing the temperature, the chemical exchange between the nonequivalent cation sites was seen on the 63Cu NMR spectra. We could determine the activation energy for this motion to be 35 kJ mol-1 , in good agreement with the published ionic conductivity.

How might the London 2012 Olympics influence health and the determinants of health? Local newspaper analysis of pre-Games pathways and impacts

PubMed Central

Selvanayagam, Marinie; Thompson, Claire; Taylor, Stephanie J C; Cummins, Steven; Bourke, Liam

2012-01-01

Objectives To conduct a content analysis of pre-Games local media coverage of the potential impact on health and the determinants of health in Newham, the site of the Olympic Park. Design Local newspaper content analysis. Setting Olympic park host site of the London Borough of Newham. Outcome measures Media coverage of employment, physical activity and well-being. Results Three hundred and 51 articles meeting the inclusion criteria were included in the analysis. The overwhelming majority of the articles took a positive perspective on the Olympic Games being hosted in Newham with less than 10% (32/351) addressing potential adverse effects. The frequency of articles reporting on both employment and well-being increased significantly over time (p=0.002 and p=0.006, respectively). A non-significant increasing trend was observed for physical activity (p=0.146). New employment opportunities and the promotion of physical activity in young people were the pathways most frequently reported in the local media. However, much less attention is devoted to understanding the uncertainties about how much of these new opportunities will directly improve the determinants of health in the Newham population. Conclusions Pre-Games reporting on the impact on health and the determinants of health increased over time in the London Borough of Newham, and is overwhelmingly positive. However, specific uncertainties around the true nature of its impact on local employment and physical activity were articulated. Further evaluation of the tangible impacts on population health, and the determinants of health and health inequalities from the London 2012 Olympics, is required. PMID:23151394

Use of a web site to increase knowledge and awareness of hunger-related issues.

PubMed Central

Jennings, Sharla; Cotugna, Nancy; Vickery, Connie E.

2003-01-01

The purpose of this study was to determine the current level of knowledge and awareness of hunger-related issues among a convenience sample of Delawareans. We also assessed whether raising knowledge and awareness of the hunger problem through the FBD's newly designed web site would encourage participation in antihunger activities. Via e-mail, 1,719 individuals were invited to participate in a three-phase, online survey, and 392 agreed. Phase-I questions were answered prior to viewing the web site, phase II (n=217) immediately afterward, and phase III (n=61) six weeks later. Responses indicated a high level of awareness about general hunger issues but specific knowledge proved to be at a lower level. No statistically significant differences were noted when data were collapsed across gender, age, educational level, or work setting. In a six-week post-survey, 41% of subjects were motivated by the web site to engage in an antihunger activity; 34% had told others about the web site and indicated it may be a useful tool in antihunger outreach efforts for the FBD. PMID:14651376

Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Low, Wai Yee; Feil, Susanne C.; Ng, Hooi Ling

2010-06-14

GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 {angstrom} resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model ofmore » the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional {sup 1}H,{sup 15}N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.« less

Identification of continuous interaction sites in PLA(2)-based protein complexes by peptide arrays.

PubMed

Fortes-Dias, Consuelo Latorre; Santos, Roberta Márcia Marques dos; Magro, Angelo José; Fontes, Marcos Roberto de Mattos; Chávez-Olórtegui, Carlos; Granier, Claude

2009-01-01

Crotoxin (CA.CB) is a beta-neurotoxin from Crotalus durissus terrificus snake venom that is responsible for main envenomation effects upon biting by this snake. It is a heterodimer of an acidic protein (CA) devoid of any biological activity per se and a basic, enzymatically active, PLA(2) counterpart (CB). Both lethal and enzymatic activities of crotoxin have been shown to be inhibited by CNF, a protein from the blood of C. d. terrificus snakes. CNF replaces CA in the CA.CB complex, forming a stable, non-toxic complex CNF.CB. The molecular sites involved in the tight interfacial protein-protein interactions in these PLA(2)-based complexes have not been clearly determined. To help address this question, we used the peptide arrays approach to map possible interfacial interaction sites in CA.CB and CNF.CB. Amino acid stretches putatively involved in these interactions were firstly identified in the primary structure of CB. Further analysis of the interfacial availability of these stretches in the presumed biologically active structure of CB, suggested two interaction main sites, located at the amino-terminus and beta-wing regions. Peptide segments at the carboxyl-terminus of CB were also suggested to play a secondary role in the binding of both CA and CNF.

Do site-specific radiocarbon measurements reflect localized distributions of 14C in biota inhabiting a wetland with point contamination sources?

PubMed

Yankovich, T; King-Sharp, K J; Benz, M L; Carr, J; Killey, R W D; Beresford, N A; Wood, M D

2013-12-01

Duke Swamp is a wetland ecosystem that receives (14)C via a groundwater pathway originating from a waste management area on Atomic Energy Canada Limited's Chalk River Laboratories site. This groundwater reaches the surface of the swamp, resulting in relatively high (14)C levels over an area of 146 m(2). The objective of this study was to quantify (14)C concentrations in flora and fauna inhabiting areas of Duke Swamp over the gradient of (14)C activity concentrations in moss to determine whether (14)C specific activities in receptor biota reflect the localized nature of the groundwater source in the swamp. Representative receptor plants and animals, and corresponding air and soil samples were collected at six sites in Duke Swamp with (14)C specific activities in air that ranged from 1140 to 45,900 Bq/kg C. In general, it was found that specific activities of (14)C in biota tissues reflected those measured in environmental media collected from the same sampling site. The findings demonstrate that mosses could be used in monitoring programs to ensure protection of biota in areas with elevated (14)C, negating the need to capture and euthanize higher organisms. Copyright © 2013 Elsevier Ltd. All rights reserved.

γ-Adducin Stimulates the Thiazide-sensitive NaCl Cotransporter

PubMed Central

Dimke, Henrik; San-Cristobal, Pedro; de Graaf, Mark; Lenders, Jacques W.; Deinum, Jaap; Hoenderop, Joost G.J.

2011-01-01

The thiazide-sensitive NaCl cotransporter (NCC) plays a key role in renal salt reabsorption and the determination of systemic BP, but the molecular mechanisms governing the regulation of NCC are not completely understood. Here, through pull-down experiments coupled to mass spectrometry, we found that γ-adducin interacts with the NCC transporter. γ-Adducin colocalized with NCC to the distal convoluted tubule. 22Na+ uptake experiments in the Xenopus laevis oocyte showed that γ-adducin stimulated NCC activity in a dose-dependent manner, an effect that occurred upstream from With No Lysine (WNK) 4 kinase. The binding site of γ-adducin mapped to the N terminus of NCC and encompassed three previously reported phosphorylation sites. Supporting this site of interaction, competition with the N-terminal domain of NCC abolished the stimulatory effect of γ-adducin on the transporter. γ-Adducin failed to increase NCC activity when these phosphorylation sites were constitutively inactive or active. In addition, γ-adducin bound only to the dephosphorylated N terminus of NCC. Taken together, our observations suggest that γ-adducin dynamically regulates NCC, likely by amending the phosphorylation state, and consequently the activity, of the transporter. These data suggest that γ-adducin may influence BP homeostasis by modulating renal NaCl transport. PMID:21164023

A computational study of the dechlorination of β-hexachlorocyclohexane (β-HCH) catalyzed by the haloalkane dehalogenase LinB.

PubMed

Manna, Rabindra Nath; Dybala-Defratyka, Agnieszka

2014-11-15

LinB, a haloalkane dehalogenase from Sphingomonas paucimobilis UT26, is known to metabolize halohydrocarbons to halide ions and the respective alcohols. Its broad substrate specificity allowed its consideration for bioremediation. Herein, we have shown its catalytic action toward β-hexachlorocyclohexane (β-HCH) - an example of large-size substrates that can be accommodated in its active site. We have analyzed the capability of combined QM/MM schemes to describe in detail the SN2 dechlorination reaction between β-HCH and Asp108 in the active site of LinB. Free energy surfaces have been calculated using one and two dimensional potentials of mean force (PMF) obtained at the PM3/MM (MM=amberff99SB, TIP3P) level of theory. The overestimated energetic barriers by the PM3 Hamiltonian were corrected using a DFT functional (M06-2X). The resulted activation energies (16 and 19 kcal mol(-1) from 1D and 2D-PMF profiles, respectively) for the dechlorination reaction of β-HCH in the active site of LinB enzyme are in qualitative agreement with the experimentally determined value of 17 kcal mol(-1). The binding of β-HCH to the active site of LinB has been compared to the binding of smaller 1-chlorobutane (1-CB) and larger δ-hexabromocyclododecane (δ-HBCD). Copyright © 2014 Elsevier Inc. All rights reserved.

Significance of Ser-188 in human mitochondrial NAD kinase as determined by phosphomimetic and phosphoresistant amino-acid substitutions.

PubMed

Kawabata, Yutaka; Murata, Kousaku; Kawai, Shigeyuki

2015-12-25

Human mitochondrial NAD kinase is a crucial enzyme responsible for the synthesis of mitochondrial NADP(+). Despite its significance, little is known about the regulation of this enzyme in the mitochondria. Several putative and known phosphorylation sites within the protein have been found using phosphoproteomics, and here, we examined the effect of phosphomimetic mutations at six of these sites. The enzymatic activity was downregulated by a substitution of an Asp residue at Ser-289 and Ser-376, but not a substitution of Ala, suggesting that the phosphorylation of these residues downregulates the enzyme. Moreover, the activity was completely inhibited by substituting Ser-188 with an Asp, Glu, or in particular Ala, which highlights two possibilities: first, that Ser-188 is critical for catalytic activity, and second, that phosphorylation of Ser-188 inhibits the activity. Ser-188, Ser-289, and Ser-376 were found to be highly conserved in the primary structures of mitochondrial NAD kinase homologs in higher animals. Moreover, Ser-188 has been frequently detected in human and mouse phosphorylation site studies, whereas Ser-289 and Ser-376 have not. Taken together, this indicates that Ser-188 (and perhaps the other residues) is an important phosphorylation site that can downregulate the NAD kinase activity of this critical enzyme. Copyright © 2015 Elsevier Inc. All rights reserved.

Structure-Based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, James A.; Wilson, Heather L.; Rajagopalan, K.V.

Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conservedmore » in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 {angstrom} resolution, respectively.« less

Noninvasive imaging of three-dimensional cardiac activation sequence during pacing and ventricular tachycardia.

PubMed

Han, Chengzong; Pogwizd, Steven M; Killingsworth, Cheryl R; He, Bin

2011-08-01

Imaging cardiac excitation within ventricular myocardium is important in the treatment of cardiac arrhythmias and might help improve our understanding of arrhythmia mechanisms. This study sought to rigorously assess the imaging performance of a 3-dimensional (3D) cardiac electrical imaging (3DCEI) technique with the aid of 3D intracardiac mapping from up to 216 intramural sites during paced rhythm and norepinephrine (NE)-induced ventricular tachycardia (VT) in the rabbit heart. Body surface potentials and intramural bipolar electrical recordings were simultaneously measured in a closed-chest condition in 13 healthy rabbits. Single-site pacing and dual-site pacing were performed from ventricular walls and septum. VTs and premature ventricular complexes (PVCs) were induced by intravenous NE. Computed tomography images were obtained to construct geometry models. The noninvasively imaged activation sequence correlated well with invasively measured counterpart, with a correlation coefficient of 0.72 ± 0.04, and a relative error of 0.30 ± 0.02 averaged over 520 paced beats as well as 73 NE-induced PVCs and VT beats. All PVCs and VT beats initiated in the subendocardium by a nonreentrant mechanism. The averaged distance from the imaged site of initial activation to the pacing site or site of arrhythmias determined from intracardiac mapping was ∼5 mm. For dual-site pacing, the double origins were identified when they were located at contralateral sides of ventricles or at the lateral wall and the apex. 3DCEI can noninvasively delineate important features of focal or multifocal ventricular excitation. It offers the potential to aid in localizing the origins and imaging activation sequences of ventricular arrhythmias, and to provide noninvasive assessment of the underlying arrhythmia mechanisms. Copyright © 2011 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Noninvasive Imaging of Three-dimensional Cardiac Activation Sequence during Pacing and Ventricular Tachycardia

PubMed Central

Han, Chengzong; Pogwizd, Steven M.; Killingsworth, Cheryl R.; He, Bin

2011-01-01

Background Imaging cardiac excitation within ventricular myocardium is important in the treatment of cardiac arrhythmias and might help improve our understanding of arrhythmia mechanisms. Objective This study aims to rigorously assess the imaging performance of a three-dimensional (3-D) cardiac electrical imaging (3-DCEI) technique with the aid of 3-D intra-cardiac mapping from up to 216 intramural sites during paced rhythm and norepinephrine (NE) induced ventricular tachycardia (VT) in the rabbit heart. Methods Body surface potentials and intramural bipolar electrical recordings were simultaneously measured in a closed-chest condition in thirteen healthy rabbits. Single-site pacing and dual-site pacing were performed from ventricular walls and septum. VTs and premature ventricular complexes (PVCs) were induced by intravenous NE. Computer tomography images were obtained to construct geometry model. Results The non-invasively imaged activation sequence correlated well with invasively measured counterparts, with a correlation coefficient of 0.72±0.04, and a relative error of 0.30±0.02 averaged over 520 paced beats as well as 73 NE-induced PVCs and VT beats. All PVCs and VT beats initiated in the subendocardium by a nonreentrant mechanism. The averaged distance from imaged site of initial activation to pacing site or site of arrhythmias determined from intra-cardiac mapping was ~5mm. For dual-site pacing, the double origins were identified when they were located at contralateral sides of ventricles or at the lateral wall and the apex. Conclusion 3-DCEI can non-invasively delineate important features of focal or multi-focal ventricular excitation. It offers the potential to aid in localizing the origins and imaging activation sequence of ventricular arrhythmias, and to provide noninvasive assessment of the underlying arrhythmia mechanisms. PMID:21397046

Federal environmental standards of potential importance to operations and activities at US Department of Energy sites. Draft

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowler, K.M.; Bilyard, G.R.; Davidson, S.A.

1993-06-01

The US Department of Energy (DOE) is now engaged in a program of environmental restoration nationwide across its 45 sites. It is also bringing its facilities into compliance with environmental regulations, decontaminating and decommissioning unwanted facilities, and constructing new waste management facilities. One of the most difficult questions that DOE must face in successfully remediating its inactive waste sites, decontaminating and decommissioning its inactive facilities, and operating its waste management facilities is: ``What criteria and standards should be met?`` Acceptable standards or procedures for determining standards will assist DOE in its conduct of ongoing waste management and pending cleanup activitiesmore » by helping to ensure that those activities are conducted in compliance with applicable laws and regulations and are accepted by the regulatory community and the public. This document reports on the second of three baseline activities that are being conducted as prerequisites to either the development of quantitative standards that could be used by DOE, or consistent procedures for developing such standards. The first and third baseline activities are also briefly discussed in conjunction with the second of the three activities.« less

Substrate-specifying determinants of the nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2

PubMed Central

2004-01-01

The nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2/autotaxin are structurally related eukaryotic ecto-enzymes, but display a very different substrate specificity. NPP1 releases nucleoside 5′-monophosphates from various nucleotides, whereas NPP2 mainly functions as a lysophospholipase D. We have used a domain-swapping approach to map substrate-specifying determinants of NPP1 and NPP2. The catalytic domain of NPP1 fused to the N- and C-terminal domains of NPP2 was hyperactive as a nucleotide phosphodiesterase, but did not show any lysophospholipase D activity. In contrast, chimaeras of the catalytic domain of NPP2 and the N- and/or C-terminal domains of NPP1 were completely inactive. These data indicate that the catalytic domain as well as both extremities of NPP2 contain lysophospholipid-specifying sequences. Within the catalytic domain of NPP1 and NPP2, we have mapped residues close to the catalytic site that determine the activities towards nucleotides and lysophospholipids. We also show that the conserved Gly/Phe-Xaa-Gly-Xaa-Xaa-Gly (G/FXGXXG) motif near the catalytic site is required for metal binding, but is not involved in substrate-specification. Our data suggest that the distinct activities of NPP1 and NPP2 stem from multiple differences throughout the polypeptide chain. PMID:15096095

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stojanoski, Vlatko; Sankaran, Banumathi; Prasad, B. V. Venkataram

Due to the paucity of novel antibiotics, colistin has become a last resort antibiotic for treating multidrug resistant bacteria. Colistin acts by binding the lipid A component of lipopolysaccharides and subsequently disrupting the bacterial membrane. The recently identified plasmid-encoded MCR-1 enzyme is the first transmissible colistin resistance determinant and is a cause for concern for the spread of this resistance trait. MCR-1 is a phosphoethanolamine transferase that catalyzes the addition of phosphoethanolamine to lipid A to decrease colistin affinity. The structure of the catalytic domain of MCR-1 at 1.32 Å reveals the active site is similar to that of relatedmore » phosphoethanolamine transferases. The putative nucleophile for catalysis, threonine 285, is phosphorylated in cMCR-1 and a zinc is present at a conserved site in addition to three zincs more peripherally located in the active site. As noted for catalytic domains of other phosphoethanolamine transferases, binding sites for the lipid A and phosphatidylethanolamine substrates are not apparent in the cMCR-1 structure, suggesting that they are present in the membrane domain.« less

Environmental impact reduction through ecological planning at Bahia Magdalena, Mexico.

PubMed

Malagrino, Giovanni; Lagunas, Magdalena; Rubio, Alfredo Ortega

2008-03-01

For analyzing basic marine and coastal characteristics we selected the potential sites where shrimp culture could be developed in a large coastal zone, Bahia Magdalena, Baja California Sur, Mexico. Based on our analysis, 6 sites were preselected and field stages of work were then developed to assess the precise suitability of each site in order to develop the proposed aquaculture activities. In ranking the suitability we were able to recommend the most appropriate places to develop shrimp culture in this region. Also, knowing the exact biological, physico-chemical and social environment, we determined the best species to cultivate, the recommended total area and the methodology to be used to lessen the environmental impact and to obtain the maximum profitability Our methodology could be used not only to select appropriate sites for shrimp culture in other coastal lagoons, but it also could be applied to assess the suitability in a quick and accurate way, of any other production activity in coastal zones.

CO{sub 2} pellet decontamination technology at Westinghouse Hanford

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aldridge, T.L.; Aldrich, L.K. II; Bowman, E.V.

1995-03-01

Experimentation and testing with CO{sub 2} pellet decontamination technology is being conducted at Westinghosue Hanford Company (WHC), Richland, Washington. There are 1,100 known existing waste sites at Hanford. The sites specified by federal and state agencies are currently being studied to determine the appropriate cleanup methods best for each site. These sites are contaminated and work on them is in compliance with the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA). There are also 63 treatment, storage, and disposal units, for example: groups of waste tanks or drums. In 1992, there were 100 planned activities scheduled to bring these unitsmore » into the Resource Conservation and Recovery Act (RCRA) compliance or close them after waste removal. Ninety-six of these were completed. The remaining four were delayed or are being negotiated with regulatory agencies. As a result of past defense program activities at Hanford a tremendous volume of materials and equipment have accumulated and require remediation.« less

An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering.

PubMed

Keedy, Daniel A; Hill, Zachary B; Biel, Justin T; Kang, Emily; Rettenmaier, T Justin; Brandao-Neto, Jose; Pearce, Nicholas M; von Delft, Frank; Wells, James A; Fraser, James S

2018-06-07

Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function. © 2018, Keedy et al.

Chloroperoxidase-catalyzed oxidation of 4,6-dimethyldibenzothiophene as dimer complexes: evidence for kinetic cooperativity.

PubMed

Torres, Eduardo; Aburto, Jorge

2005-05-15

A sigmoidal kinetic behavior of chloroperoxidase for the oxidation of 4,6-dimethyldibenzothiophene (4,6-DMDBT) in water-miscible organic solvent is for the first time reported. Kinetics of 4,6-DMDBT oxidation showed a cooperative profile probably due to the capacity of chloroperoxidase to recognize a substrate dimer (pi-pi dimer) in its active site. Experimental evidence is given for dimer formation and its presence in the active site of chloroperoxidase. The kinetic data were adjusted for a binding site able to interact with either monomer or dimer substrates, producing a cooperative model describing a one-site binding of two related species. Determination of kinetics constants by iterative calculations of possible oxidation paths of 4,6-DMDBT suggests that kinetics oxidation of dimer substrate is preferred when compared to monomer oxidation. Steady-state fluorometry of substrate in the absence and presence of chloroperoxidase, described by the spectral center of mass, supports this last conclusion.

Determining the binding affinities of phenolic compounds to proteins by quenching of the intrinsic tryptophan fluorescence.

PubMed

Rawel, Harshadrai M; Frey, Simone K; Meidtner, Karina; Kroll, Jürgen; Schweigert, Florian J

2006-08-01

The noncovalent binding of selected phenolic compounds (chlorogenic-, ferulic-, gallic acid, quercetin, rutin, and isoquercetin) to proteins (HSA, BSA, soy glycinin, and lysozyme) was studied by an indirect method applying the quenching of intrinsic tryptophan fluorescence. From the data obtained, the binding constants were calculated by nonlinear regression (one site binding; y = Bx/k + x). It has been reported that tannins inhibit human salivary amylase and that these complexes may reduce the development of cariogenic plaques. Further, amylase contains two tryptophan residues in its active site. Therefore, in a second part of the study involving 31 human subjects, evidence was sought for noncovalent interactions between the phenols of green tea and saliva proteins as measured by the fluorescence intensity. Amylase activity was determined before and after the addition of green tea to saliva of 31 subjects. Forty percent of the subjects showed an increase in amylase activity contrary to studies reporting only a decrease in activity. The interactions of tannin with amylase result in a decrease of its activity. It still remains to be elucidated why amylase does not react uniformly under conditions of applying green tea to saliva. Further, in terms of using phenols as caries inhibitors this finding should be of importance.

Leaving Group Ability Observably Affects Transition State Structure in a Single Enzyme Active Site.

PubMed

Roston, Daniel; Demapan, Darren; Cui, Qiang

2016-06-15

A reaction's transition state (TS) structure plays a critical role in determining reactivity and has important implications for the design of catalysts, drugs, and other applications. Here, we explore TS structure in the enzyme alkaline phosphatase using hybrid Quantum Mechanics/Molecular Mechanics simulations. We find that minor perturbations to the substrate have major effects on TS structure and the way the enzyme stabilizes the TS. Substrates with good leaving groups (LGs) have little cleavage of the phosphorus-LG bond at the TS, while substrates with poor LGs have substantial cleavage of that bond. The results predict nonlinear free energy relationships for a single rate-determining step, and substantial differences in kinetic isotope effects for different substrates; both trends were observed in previous experimental studies, although the original interpretations differed from the present model. Moreover, due to different degrees of phosphorus-LG bond cleavage at the TS for different substrates, the LG is stabilized by different interactions at the TS: while a poor LG is directly stabilized by an active site zinc ion, a good LG is mainly stabilized by active site water molecules. Our results demonstrate the considerable plasticity of TS structure and stabilization in enzymes. Furthermore, perturbations to reactivity that probe TS structure experimentally (i.e., substituent effects) may substantially perturb the TS they aim to probe, and thus classical experimental approaches such as free energy relations should be interpreted with care.

Structural Determinants of Autoproteolysis of the Haemophilus influenzae Hap Autotransporter▿

PubMed Central

Kenjale, Roma; Meng, Guoyu; Fink, Doran L.; Juehne, Twyla; Ohashi, Tomoo; Erickson, Harold P.; Waksman, Gabriel; St. Geme, Joseph W.

2009-01-01

Haemophilus influenzae is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hap autotransporter protein mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells and presumably facilitates colonization. The serine protease activity of Hap is associated with autoproteolytic cleavage and extracellular release of the HapS passenger domain, leaving the Hapβ C-terminal domain embedded in the outer membrane. Cleavage occurs most efficiently at the LN1036-37 peptide bond and to a lesser extent at three other sites. In this study, we utilized site-directed mutagenesis, homology modeling, and assays with a peptide library to characterize the structural determinants of Hap proteolytic activity and cleavage specificity. In addition, we used homology modeling to predict the S1, S2, and S4 subsite residues of the Hap substrate groove. Our results indicate that the P1 and P2 positions at the Hap cleavage sites are critical for cleavage, with leucine preferred over larger hydrophobic residues or other amino acids in these positions. The substrate groove is formed by L263 and N274 at the S1 subsite, R264 at the S2 subsite, and E265 at the S4 subsite. This information may facilitate design of approaches to block Hap activity and interfere with H. influenzae colonization. PMID:19687208

The AMPA receptor subunit GluR1 regulates dendritic architecture of motor neurons

NASA Technical Reports Server (NTRS)

Inglis, Fiona M.; Crockett, Richard; Korada, Sailaja; Abraham, Wickliffe C.; Hollmann, Michael; Kalb, Robert G.

2002-01-01

The morphology of the mature motor neuron dendritic arbor is determined by activity-dependent processes occurring during a critical period in early postnatal life. The abundance of the AMPA receptor subunit GluR1 in motor neurons is very high during this period and subsequently falls to a negligible level. To test the role of GluR1 in dendrite morphogenesis, we reintroduced GluR1 into rat motor neurons at the end of the critical period and quantitatively studied the effects on dendrite architecture. Two versions of GluR1 were studied that differed by the amino acid in the "Q/R" editing site. The amino acid occupying this site determines single-channel conductance, ionic permeability, and other essential electrophysiologic properties of the resulting receptor channels. We found large-scale remodeling of dendritic architectures in a manner depending on the amino acid occupying the Q/R editing site. Alterations in the distribution of dendritic arbor were not prevented by blocking NMDA receptors. These observations suggest that the expression of GluR1 in motor neurons modulates a component of the molecular substrate of activity-dependent dendrite morphogenesis. The control of these events relies on subunit-specific properties of AMPA receptors.

Active Faults and Earthquake Hazards in the FY 79 Verification Sites - Nevada-Utah Siting Region.

DTIC Science & Technology

1980-03-26

structures, such as shelters and command/control facilities, away from rup- ture hazards. Again, the probability of rupture, the effect of damage and ...accommodate an MCE, and less critical structures (such as the shelters ) designed for a probabilistically determined event, may have merit for the MX...B., and Eaton, G. P., eds., Cenozoic tectonics and regional geophysics of the western cordillera : Geol. Soc. Am. Mem. 152, p. 1-32. Stewart, J. H

The structure and function of supported molybdenum nitride and molybdenum carbide hydrotreating catalysts

NASA Astrophysics Data System (ADS)

Dolce, Gregory Martin

1997-11-01

A series of gamma-Alsb2Osb3 supported molybdenum nitrides and carbides were prepared by the temperature programmed reaction of supported molybdates with ammonia and methane/hydrogen mixtures, respectively. In the first part of this research, the effects of synthesis heating rates and molybdenum loading on the catalytic properties of the materials were examined. A significant amount of excess carbon was deposited on the surface of the carbides during synthesis. The materials consisted of small particles which were very highly dispersed. Oxygen chemisorption indicated that the nitride particles may have been two-dimensional. The dispersion of the carbides, however, appeared to decrease as the loading increased. The catalysts were evaluated for hydrodenitrogenation (HDN), hydrodesulfurization (HDS), and hydrodeoxygenation (HDO). The molybdenum loading had the largest effect on the activity of the materials. For the nitrides, the HDN and HDS activities were inverse functions of the loading. This suggested that the most active HDN and HDS sites were located at the perimeter of the two-dimensional particles. The HDN and HDS activities of the carbides followed the same trend as the oxygen uptake. This result suggested that oxygen titrated the active sites on the supported carbides. Selected catalysts were evaluated for methylcarbazole HDN, dibenzothiophene HDS, and dibenzofuran HDO. The activity and selectivity of the nitrides and carbides were competitive with a presulfided commercial catalyst. In the second part of this work, a series of supported nitrides and carbides were prepared using a wider range of loadings (5-30 wt% Mo). Thermogravimetric analysis was used to determine the temperature at which excess carbon was deposited on the carbides. By modifying the synthesis parameters, the deposition of excess carbon was effectively inhibited. The dispersions of the supported nitrides and carbides were constant and suggested that the materials consisted of two-dimensional raft-like particles. The HDN activity of the nitrides decreased as the loading increased, while that of the carbides was relatively constant. Carbon monoxide and methylamine adsorbed on the same types of sites on the nitrides and carbides. Infrared spectroscopy and temperature programmed desorption revealed that some methylamine underwent HDN on the nitrides and carbides. Carbon monoxide appeared to adsorb on two types of sites. One type of site adsorbed CO which desorbed upon heating while the other type of site adsorbed CO which dissociated when the material was heated. The relative amounts of desorbed CO and methylamine scaled with the activity of the nitrides suggesting that CO and methylamine titrated the active sites. It appeared that the active sites of the supported carbides were different from those on the supported nitrides. It was proposed that the active sites on the supported nitrides were at the perimeter of the two-dimensional particles while the active sites of the carbides were "on top" of the particles.

Hydrologic data collection activities in the Solomon Gulch basin near Valdez, Alaska

USGS Publications Warehouse

Bigelow, B.B.

1988-01-01

In 1981, the Alaska Power Authority completed construction of a dam spillway at the north end of Solomon Lake near Valdez. Regulation and diversion from the dam since 1982 have significantly altered the natural flow characteristics of Solomon Gulch. In September 1986, the Geological Survey began data collection to determine mean daily discharge at four sites below Solomon Lake and thus document the effects of regulation and diversion of water on the flow at various points in the system. Periodic discharge measurements and continuous records of water stage were obtained at two of the sites, and daily discharge values were computed for all four sites. The report contains descriptions of the data collection sites and the discharge records for each site for September 1986 and water year 1987. (USGS)

Determining rotational dynamics of the guanidino group of arginine side chains in proteins by carbon-detected NMR.

PubMed

Gerecht, Karola; Figueiredo, Angelo Miguel; Hansen, D Flemming

2017-09-16

Arginine residues are imperative for many active sites and protein-interaction interfaces. A new NMR-based method is presented to determine the rotational dynamics around the N ε -C ζ bond of arginine side chains. An application to a 19 kDa protein shows that the strengths of interactions involving arginine side chains can be characterised.

Arginine residues on the opposite side of the active site stimulate the catalysis of ribosome depurination by ricin A chain by interacting with the P-protein stalk.

PubMed

Li, Xiao-Ping; Kahn, Peter C; Kahn, Jennifer Nielsen; Grela, Przemyslaw; Tumer, Nilgun E

2013-10-18

Ricin inhibits protein synthesis by depurinating the α-sarcin/ricin loop (SRL). Ricin holotoxin does not inhibit translation unless the disulfide bond between the A (RTA) and B (RTB) subunits is reduced. Ricin holotoxin did not bind ribosomes or depurinate them but could depurinate free RNA. When RTA is separated from RTB, arginine residues located at the interface are exposed to the solvent. Because this positively charged region, but not the active site, is blocked by RTB, we mutated arginine residues at or near the interface of RTB to determine if they are critical for ribosome binding. These variants were structurally similar to wild type RTA but could not bind ribosomes. Their K(m) values and catalytic rates (k(cat)) for an SRL mimic RNA were similar to those of wild type, indicating that their activity was not altered. However, they showed an up to 5-fold increase in K(m) and up to 38-fold decrease in kcat toward ribosomes. These results suggest that the stalk binding stimulates the catalysis of ribosome depurination by RTA. The mutated arginines have side chains behind the active site cleft, indicating that the ribosome binding surface of RTA is on the opposite side of the surface that interacts with the SRL. We propose that stalk binding stimulates the catalysis of ribosome depurination by orienting the active site of RTA toward the SRL and thereby allows docking of the target adenine into the active site. This model may apply to the translation factors that interact with the stalk.

PqsBC, a Condensing Enzyme in the Biosynthesis of the Pseudomonas aeruginosa Quinolone Signal

PubMed Central

Drees, Steffen Lorenz; Li, Chan; Prasetya, Fajar; Saleem, Muhammad; Dreveny, Ingrid; Williams, Paul; Hennecke, Ulrich; Emsley, Jonas; Fetzner, Susanne

2016-01-01

Pseudomonas aeruginosa produces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used for in vitro binding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis. PMID:26811339

Bacterial abundance and activity in deep-sea sediments from the eastern North Atlantic

NASA Astrophysics Data System (ADS)

Eardly, D. F.; Carton, M. W.; Gallagher, J. M.; Patching, J. W.

Results are presented from four cruises to the Porcupine Abyssal Plain (PAP site) that took place during the BENGAL project from September 1996 to March 1998, and two cruises to the PAP and an oligotrophic site (EUMELI) that took place during the DEEPSEAS project between September 1993 and March 1994. Bacterial abundances in sediment and sediment contact water were measured by epifluorescence microscopy. Bacterial activity was determined by 3H-thymidine incorporation as a measure of DNA synthesis, and by 3H-leucine incorporation as a measure of protein synthesis. Activities were measured under atmospheric and in situ pressures and temperatures. Bacterial activity was usually higher in samples incubated at in situ pressure than those incubated at atmospheric pressure indicating that a barophilic community was dominant. Inter-cruise comparisons of abundance and activity during the BENGAL project showed no firm evidence of there being a seasonal response in the benthic microbial community to any episodic phytodetritus event. This was probably because of inter-annual variations in the quality and quantity of phytodetritus deposition at the PAP site, the rapid remineralization of fresh organic material by the microbial communities and the timing of cruises to the study area. 3H-thymidine and 3H-leucine incorporation in sediments was higher during the BENGAL period than the DEEPSEAS programme. A methodological change in the 3H-thymidine incorporation technique for sediments may explain the differences in DNA synthesis observed between the two projects, whereas the lower levels of protein synthesis observed during the DEEPSEAS programme probably reflected both inter-annual variations in activity at the PAP site and the lower productivity that prevailed at surface at the EUMELI oligotrophic site. Rates of 3H-thymidine incorporation in sediment contact water were similar during both projects.

XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs

PubMed Central

Scott, Fiona L; Denault, Jean-Bernard; Riedl, Stefan J; Shin, Hwain; Renatus, Martin; Salvesen, Guy S

2005-01-01

The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. We used site-directed mutagenesis of BIR2 and its linker to determine the mechanism of executioner caspase inhibition by XIAP. We show that the BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, we predict that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP. PMID:15650747

Free and ATP-bound structures of Ap4A hydrolase from Aquifex aeolicus V5.

PubMed

Jeyakanthan, Jeyaraman; Kanaujia, Shankar Prasad; Nishida, Yuya; Nakagawa, Noriko; Praveen, Surendran; Shinkai, Akeo; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Sekar, Kanagaraj

2010-02-01

Asymmetric diadenosine tetraphosphate (Ap(4)A) hydrolases degrade the metabolite Ap(4)A back into ATP and AMP. The three-dimensional crystal structure of Ap(4)A hydrolase (16 kDa) from Aquifex aeolicus has been determined in free and ATP-bound forms at 1.8 and 1.95 A resolution, respectively. The overall three-dimensional crystal structure of the enzyme shows an alphabetaalpha-sandwich architecture with a characteristic loop adjacent to the catalytic site of the protein molecule. The ATP molecule is bound in the primary active site and the adenine moiety of the nucleotide binds in a ring-stacking arrangement equivalent to that observed in the X-ray structure of Ap(4)A hydrolase from Caenorhabditis elegans. Binding of ATP in the active site induces local conformational changes which may have important implications in the mechanism of substrate recognition in this class of enzymes. Furthermore, two invariant water molecules have been identified and their possible structural and/or functional roles are discussed. In addition, modelling of the substrate molecule at the primary active site of the enzyme suggests a possible path for entry and/or exit of the substrate and/or product molecule.

Bacterial Abundance and Activity across Sites within Two Northern Wisconsin Sphagnum Bogs.

PubMed

Fisher; Graham; Graham

1998-11-01

Abstract Bacterial abundance, temperature, pH, and dissolved organic carbon (DOC) concentration were compared across surface sites within and between two northern Wisconsin Sphagnum peatlands over the summer seasons in 1995 and 1996. Sites of interest were the Sphagnum mat surface, the water-filled moat (lagg) at the bog margin, and the bog lake littoral zone. Significant differences in both bacterial populations and water chemistry were observed between sites. pH was highest in the lake and lowest in the mat at both bogs; the opposite was true for DOC. Large populations of bacteria were present in surface interstitial water from the mat; abundance in this site was consistently higher than in the moat or lake. Bacterial abundance also increased across sites of increasing DOC concentration and declining pH. Bacterial activities (rates of [3H]leucine incorporation) and growth in dilution cultures (with grazers removed) were also assessed in lake, moat, and mat sites. Results using these measures generally supported the trends observed in abundance, although high rates of [3H]leucine incorporation were recorded in the moat at one of the bogs. Our results indicate that bacterial populations in Sphagnum peatlands are not adversely affected by acidity, and that DOC may be more important than pH in determining bacterial abundance in these environments.

Loggerhead sea turtle environmental sex determination: implications of moisture and temperature for climate change based predictions for species survival.

PubMed

Wyneken, Jeanette; Lolavar, Alexandra

2015-05-01

It has been proposed that because marine turtles have environmentally determined sex by incubation temperature, elevated temperatures might skew sex ratios to unsustainable levels, leading to extinction. Elevated temperatures may also reduce availability of suitable nesting sites via sea level rise. Increased tropical storm activity can directly affect nest site moisture, embryonic development, and the probability that nests will survive. Here, we question some of these assumptions and review the limits of sex ratio estimates. Sea turtles may be more resilient to climate change than previously thought, in part because of hitherto unappreciated mechanisms for coping with variable incubation conditions. © 2015 Wiley Periodicals, Inc.

Global evaluation and calibration of a passive air sampler for gaseous mercury

NASA Astrophysics Data System (ADS)

McLagan, David S.; Mitchell, Carl P. J.; Steffen, Alexandra; Hung, Hayley; Shin, Cecilia; Stupple, Geoff W.; Olson, Mark L.; Luke, Winston T.; Kelley, Paul; Howard, Dean; Edwards, Grant C.; Nelson, Peter F.; Xiao, Hang; Sheu, Guey-Rong; Dreyer, Annekatrin; Huang, Haiyong; Hussain, Batual Abdul; Lei, Ying D.; Tavshunsky, Ilana; Wania, Frank

2018-04-01

Passive air samplers (PASs) for gaseous mercury (Hg) were deployed for time periods between 1 month and 1 year at 20 sites across the globe with continuous atmospheric Hg monitoring using active Tekran instruments. The purpose was to evaluate the accuracy of the PAS vis-à-vis the industry standard active instruments and to determine a sampling rate (SR; the volume of air stripped of gaseous Hg per unit of time) that is applicable across a wide range of conditions. The sites spanned a wide range of latitudes, altitudes, meteorological conditions, and gaseous Hg concentrations. Precision, based on 378 replicated deployments performed by numerous personnel at multiple sites, is 3.6 ± 3.0 %1, confirming the PAS's excellent reproducibility and ease of use. Using a SR previously determined at a single site, gaseous Hg concentrations derived from the globally distributed PASs deviate from Tekran-based concentrations by 14.2 ± 10 %. A recalibration using the entire new data set yields a slightly higher SR of 0.1354 ± 0.016 m3 day-1. When concentrations are derived from the PAS using this revised SR the difference between concentrations from active and passive sampling is reduced to 8.8 ± 7.5 %. At the mean gaseous Hg concentration across the study sites of 1.54 ng m-3, this represents an ability to resolve concentrations to within 0.13 ng m-3. Adjusting the sampling rate to deployment specific temperatures and wind speeds does not decrease the difference in active-passive concentration further (8.7 ± 5.7 %), but reduces its variability by leading to better agreement in Hg concentrations measured at sites with very high and very low temperatures and very high wind speeds. This value (8.7 ± 5.7 %) represents a conservative assessment of the overall uncertainty of the PAS due to inherent uncertainties of the Tekran instruments. Going forward, the recalibrated SR adjusted for temperature and wind speed should be used, especially if conditions are highly variable or deviate considerably from the average of the deployments in this study (9.89 °C, 3.41 m s-1). Overall, the study demonstrates that the sampler is capable of recording background gaseous Hg concentrations across a wide range of environmental conditions with accuracy similar to that of industry standard active sampling instruments. Results at sites with active speciation units were inconclusive on whether the PASs take up total gaseous Hg or solely gaseous elemental Hg primarily because gaseous oxidized Hg concentrations were in a similar range as the uncertainty of the PAS. 1Subscripted numbers are not significant, but are reported to reduce rounding errors in subsequent studies (see Sect. 2.3 for details).

Structural studies of viperin, an antiviral radical SAM enzyme.

PubMed

Fenwick, Michael K; Li, Yue; Cresswell, Peter; Modis, Yorgo; Ealick, Steven E

2017-06-27

Viperin is an IFN-inducible radical S -adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S -adenosylhomocysteine (SAH) or 5'-deoxyadenosine (5'-dAdo) and l-methionine (l-Met). Viperin contains a partial (βα) 6 -barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first β-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5'-dAdo and l-Met (SAM cleavage products) is consistent with the canonical mechanism of 5'-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTP-binding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.

LFP Oscillations in the Mesencephalic Locomotor Region during Voluntary Locomotion

PubMed Central

Noga, Brian R.; Sanchez, Francisco J.; Villamil, Luz M.; O’Toole, Christopher; Kasicki, Stefan; Olszewski, Maciej; Cabaj, Anna M.; Majczyński, Henryk; Sławińska, Urszula; Jordan, Larry M.

2017-01-01

Oscillatory rhythms in local field potentials (LFPs) are thought to coherently bind cooperating neuronal ensembles to produce behaviors, including locomotion. LFPs recorded from sites that trigger locomotion have been used as a basis for identification of appropriate targets for deep brain stimulation (DBS) to enhance locomotor recovery in patients with gait disorders. Theta band activity (6–12 Hz) is associated with locomotor activity in locomotion-inducing sites in the hypothalamus and in the hippocampus, but the LFPs that occur in the functionally defined mesencephalic locomotor region (MLR) during locomotion have not been determined. Here we record the oscillatory activity during treadmill locomotion in MLR sites effective for inducing locomotion with electrical stimulation in rats. The results show the presence of oscillatory theta rhythms in the LFPs recorded from the most effective MLR stimulus sites (at threshold ≤60 μA). Theta activity increased at the onset of locomotion, and its power was correlated with the speed of locomotion. In animals with higher thresholds (>60 μA), the correlation between locomotor speed and theta LFP oscillations was less robust. Changes in the gamma band (previously recorded in vitro in the pedunculopontine nucleus (PPN), thought to be a part of the MLR) were relatively small. Controlled locomotion was best achieved at 10–20 Hz frequencies of MLR stimulation. Our results indicate that theta and not delta or gamma band oscillation is a suitable biomarker for identifying the functional MLR sites. PMID:28579945

Variability in carbon dioxide fluxes for dense urban, suburban and woodland environments in southern England

NASA Astrophysics Data System (ADS)

Ward, Helen; Kotthaus, Simone; Grimmond, C. Sue; Bjorkegren, Alex; Wilkinson, Matt; Morrison, Will; Evans, Jon; Morison, James; Christen, Andreas

2014-05-01

The net exchange of carbon dioxide between the surface and atmosphere can be measured using the eddy covariance technique. Fluxes from a dense urban environment (central London), a suburban landscape (Swindon) and a woodland ecosystem (Alice Holt) are compared. All sites are located in southern England and experience similar climatic and meteorological conditions, yet have very different land cover. The signatures of anthropogenic and biogenic processes are explored at various (daily, seasonal and annual) timescales. Particular emphasis is placed on identifying the mixture of controls that determine the flux. In summer, there are clear similarities between the suburban and woodland sites, as the diurnal behaviour is dominated by photosynthetic uptake. In winter, however, vegetation is largely dormant and human activity determines the pattern of fluxes at the urban and suburban sites. Emissions from building heating augment the net release of carbon dioxide in cold months. Road use is a major contributor to the total emissions, and the diurnal cycle in the observed fluxes reflects this: in central London roads are busy throughout the day, whereas in Swindon a double-peaked rush-hour signal is evident. The net exchange of carbon dioxide is estimated for each site and set in context with other studies around the world. Central London has the smallest proportion of vegetation and largest emissions amongst study sites in the literature to date. Although Swindon's appreciable vegetation fraction helps to offset the anthropogenic emissions, even in summertime the 24h total flux is usually positive, indicating carbon release. Comparison of these three sites in a similar region demonstrates the effects of increasing urban density and changing land use on the atmosphere. Findings are relevant in terms of characterising the behaviour of urban surfaces and for quantifying the impact of anthropogenic activities.

Polycyclic aromatic hydrocarbon exposure in Steller's eiders (Polysticta stelleri) and harlequin ducks (Histronicus histronicus) in the Eastern Aleutian Islands, Alaska, USA

USGS Publications Warehouse

Miles, A.K.; Flint, Paul L.; Trust, K.A.; Ricca, M.A.; Spring, S.E.; Arrieta, D.E.; Hollmen, T.; Wilson, B.W.

2007-01-01

Seaducks may be affected by harmful levels of polycyclic aromatic hydrocarbons (PAHs) at seaports near the Arctic. As an indicator of exposure to PAHs, we measured hepatic enzyme 7-ethoxyresorufin-O-deethylase activity (EROD) to determine cytochrome P4501A induction in Steller's eiders (Polysticta stelleri) and Harlequin ducks (Histronicus histronicus) from Unalaska, Popof, and Unga Islands (AK, USA) in 2002 and 2003. We measured PAHs and organic contaminants in seaduck prey samples and polychlorinated biphenyl congeners in seaduck blood plasma to determine any relationship to EROD. Using Akaike's information criterion, species and site differences best explained EROD patterns: Activity was higher in Harlequin ducks than in Steller's eiders and higher at industrial than at nonindustrial sites. Site-specific concentrations of PAHs in blue mussels ([Mytilus trossilus] seaduck prey; PAH concentrations higher at Dutch Harbor, Unalaska, than at other sites) also was important in defining EROD patterns. Organochlorine compounds rarely were detected in prey samples. No relationship was found between polychlorinated biphenyl congeners in avian blood and EROD, which further supported inferences derived from Akaike's information criterion. Congeners were highest in seaducks from a nonindustrial or reference site, contrary to PAH patterns. To assist in interpreting the field study, 15 captive Steller's eiders were dosed with a PAH known to induce cytochrome P4501A. Dosed, captive Steller's eiders had definitive induction, but results indicated that wild Steller's eiders were exposed to PAHs or other inducing compounds at levels greater than those used in laboratory studies. Concentrations of PAHs in blue mussels at or near Dutch Harbor (∼1,180–5,980 ng/g) approached those found at highly contaminated sites (∼4,100–7,500 ng/g).

How Accurate Is Your Activity Tracker? A Comparative Study of Step Counts in Low-Intensity Physical Activities.

PubMed

Alinia, Parastoo; Cain, Chris; Fallahzadeh, Ramin; Shahrokni, Armin; Cook, Diane; Ghasemzadeh, Hassan

2017-08-11

As commercially available activity trackers are being utilized in clinical trials, the research community remains uncertain about reliability of the trackers, particularly in studies that involve walking aids and low-intensity activities. While these trackers have been tested for reliability during walking and running activities, there has been limited research on validating them during low-intensity activities and walking with assistive tools. The aim of this study was to (1) determine the accuracy of 3 Fitbit devices (ie, Zip, One, and Flex) at different wearing positions (ie, pants pocket, chest, and wrist) during walking at 3 different speeds, 2.5, 5, and 8 km/h, performed by healthy adults on a treadmill; (2) determine the accuracy of the mentioned trackers worn at different sites during activities of daily living; and (3) examine whether intensity of physical activity (PA) impacts the choice of optimal wearing site of the tracker. We recruited 15 healthy young adults to perform 6 PAs while wearing 3 Fitbit devices (ie, Zip, One, and Flex) on their chest, pants pocket, and wrist. The activities include walking at 2.5, 5, and 8 km/h, pushing a shopping cart, walking with aid of a walker, and eating while sitting. We compared the number of steps counted by each tracker with gold standard numbers. We performed multiple statistical analyses to compute descriptive statistics (ie, ANOVA test), intraclass correlation coefficient (ICC), mean absolute error rate, and correlation by comparing the tracker-recorded data with that of the gold standard. All the 3 trackers demonstrated good-to-excellent (ICC>0.75) correlation with the gold standard step counts during treadmill experiments. The correlation was poor (ICC<0.60), and the error rate was significantly higher in walker experiment compared to other activities. There was no significant difference between the trackers and the gold standard in the shopping cart experiment. The wrist worn tracker, Flex, counted several steps when eating (P<.01). The chest tracker was identified as the most promising site to capture steps in more intense activities, while the wrist was the optimal wearing site in less intense activities. This feasibility study focused on 6 PAs and demonstrated that Fitbit trackers were most accurate when walking on a treadmill and least accurate during walking with a walking aid and for low-intensity activities. This may suggest excluding participants with assistive devices from studies that focus on PA interventions using commercially available trackers. This study also indicates that the wearing site of the tracker is an important factor impacting the accuracy performance. A larger scale study with a more diverse population, various activity tracker vendors, and a larger activity set are warranted to generalize our results. ©Parastoo Alinia, Chris Cain, Ramin Fallahzadeh, Armin Shahrokni, Diane Cook, Hassan Ghasemzadeh. Originally published in JMIR Mhealth and Uhealth (http://mhealth.jmir.org), 11.08.2017.

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. © 2015 Zorrilla de San Martin et al.

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

Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID.

PubMed

Wang, Meng; Rada, Cristina; Neuberger, Michael S

2010-01-18

High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide. As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine. Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots. However, AID-catalyzed deamination of IgV targets in vitro does not yield the same degree of hotspot dominance to that observed in vivo, indicating the importance of features beyond AID's active site and DNA local sequence environment in determining in vivo hotspot dominance.

The enzymatic activity of CEM15/Apobec-3G is essential for the regulation of the infectivity of HIV-1 virion but not a sole determinant of its antiviral activity.

PubMed

Shindo, Keisuke; Takaori-Kondo, Akifumi; Kobayashi, Masayuki; Abudu, Aierken; Fukunaga, Keiko; Uchiyama, Takashi

2003-11-07

Human immunodeficiency virus, type 1 (HIV-1) Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion. Vif functions to counteract an anti-HIV-1 cellular factor in non-permissive cells, CEM15/Apobec-3G, which shares a cytidine deaminase motif. CEM15/Apobec-3G deaminates dC to dU in the minus strand DNA of HIV-1, resulting in G to A hypermutation in the plus strand DNA. In this study, we have done the mutagenesis analysis on two cytidine deaminase motifs in CEM15/Apobec-3G and examined their antiviral functions as well as the DNA editing activity. Point mutations in the C-terminal active site such as E259Q and C291A almost completely abrogated the antiviral function, while those in the N-terminal active site such as E67Q and C100A retained this activity to a lesser extent as compared with that of the wild type. The DNA editing activities of E67Q and E259Q mutants were both retained but impaired to the same extent. This indicates that the enzymatic activity of this protein is essential but not a sole determinant of the antiviral activity. Furthermore, all the deletion mutants tested in this study lost the antiviral activity because of the loss of the activity for dimerization, suggesting that the entire protein structure is necessary for the antiviral function.

Dual allosteric activation mechanisms in monomeric human glucokinase

PubMed Central

Whittington, A. Carl; Larion, Mioara; Bowler, Joseph M.; Ramsey, Kristen M.; Brüschweiler, Rafael; Miller, Brian G.

2015-01-01

Cooperativity in human glucokinase (GCK), the body’s primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme’s small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the 1H-13C heteronuclear multiple quantum coherence (HMQC) spectrum of 13C-Ile–labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the 1H-13C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems. PMID:26283387

Dual allosteric activation mechanisms in monomeric human glucokinase.

PubMed

Whittington, A Carl; Larion, Mioara; Bowler, Joseph M; Ramsey, Kristen M; Brüschweiler, Rafael; Miller, Brian G

2015-09-15

Cooperativity in human glucokinase (GCK), the body's primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme's small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) spectrum of (13)C-Ile-labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the (1)H-(13)C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.

Exoenzyme activity in contaminated soils before and after soil washing: ß-glucosidase activity as a biological indicator of soil health.

PubMed

Chae, Yooeun; Cui, Rongxue; Woong Kim, Shin; An, Gyeonghyeon; Jeong, Seung-Woo; An, Youn-Joo

2017-01-01

It is essential to remediate or amend soils contaminated with various heavy metals or pollutants so that the soils may be used again safely. Verifying that the remediated or amended soils meet soil quality standards is an important part of the process. We estimated the activity levels of eight soil exoenzymes (acid phosphatase, arylsulfatase, catalase, dehydrogenase, fluorescein diacetate hydrolase, protease, urease, and ß-glucosidase) in contaminated and remediated soils from two sites near a non-ferrous metal smelter, using colorimetric and titrimetric determination methods. Our results provided the levels of activity of soil exoenzymes that indicate soil health. Most enzymes showed lower activity levels in remediated soils than in contaminated soils, with the exception of protease and urease, which showed higher activity after remediation in some soils, perhaps due to the limited nutrients available in remediated soils. Soil exoenzymes showed significantly higher activity in soils from one of the sites than from the other, due to improper conditions at the second site, including high pH, poor nutrient levels, and a high proportion of sand in the latter soil. Principal component analysis revealed that ß-glucosidase was the best indicator of soil ecosystem health, among the enzymes evaluated. We recommend using ß-glucosidase enzyme activity as a prior indicator in estimating soil ecosystem health. Copyright © 2016 Elsevier Inc. All rights reserved.

Interaction of the Transcription Start Site Core Region and Transcription Factor YY1 Determine Ascorbate Transporter SVCT2 Exon 1a Promoter Activity

PubMed Central

Qiao, Huan; May, James M.

2012-01-01

Transcription of the ascorbate transporter, SVCT2, is driven by two distinct promoters in exon 1 of the transporter sequence. The exon 1a promoter lacks a classical transcription start site and little is known about regulation of promoter activity in the transcription start site core (TSSC) region. Here we present evidence that the TSSC binds the multifunctional initiator-binding protein YY1. Electrophoresis shift assays using YY1 antibody showed that YY1 is present as one of two major complexes that specifically bind to the TSSC. The other complex contains the transcription factor NF-Y. Mutations in the TSSC that decreased YY1 binding also impaired the exon 1a promoter activity despite the presence of an upstream activating NF-Y/USF complex, suggesting that YY1 is involved in the regulation of the exon 1a transcription. Furthermore, YY1 interaction with NF-Y and/or USF synergistically enhanced the exon 1a promoter activity in transient transfections and co-activator p300 enhanced their synergistic activation. We propose that the TSSC plays a vital role in the exon 1a transcription and that this function is partially carried out by the transcription factor YY1. Moreover, co-activator p300 might be able to synergistically enhance the TSSC function via a “bridge” mechanism with upstream sequences. PMID:22532872

The molecular basis of the effect of temperature on enzyme activity.

PubMed

Daniel, Roy M; Peterson, Michelle E; Danson, Michael J; Price, Nicholas C; Kelly, Sharon M; Monk, Colin R; Weinberg, Cristina S; Oudshoorn, Matthew L; Lee, Charles K

2009-12-23

Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. The temperature midpoint (Teq) of the rapid equilibration between the two forms is related to the growth temperature of the organism, and the enthalpy of the equilibrium (DeltaHeq) to its ability to function over various temperature ranges. In the present study, we show that the difference between the active and inactive forms is at the enzyme active site. The results reveal an apparently universal mechanism, independent of enzyme reaction or structure, based at or near the active site, by which enzymes lose activity as temperature rises, as opposed to denaturation which is global. Results show that activity losses below Teq may lead to significant errors in the determination of DeltaG*cat made on the basis of the two-state ('Classical') model, and the measured kcat will then not be a true indication of an enzyme's catalytic power. Overall, the results provide a molecular rationale for observations that the active site tends to be more flexible than the enzyme as a whole, and that activity losses precede denaturation, and provide a general explanation in molecular terms for the effect of temperature on enzyme activity.

Rates and determinants of uptake and use of an internet physical activity and weight management program in office and manufacturing work sites in England: Cohort Study

USDA-ARS?s Scientific Manuscript database

Internet-based physical activity (PA) and weight management programs have the potential to improve employees' health in large occupational health settings. To be successful, the program must engage a wide range of employees, especially those at risk of weight gain or ill health. The aim of the study...

Crystal structure of the unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate.

PubMed Central

Zhang, K. Y.; Cascio, D.; Eisenberg, D.

1994-01-01

The crystal structure of unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, was determined to 2.7 A resolution by X-ray crystallography. The transition state analog binds at the active site in an extended conformation. As compared to the binding of the same analog in the activated enzyme, the analog binds in a reverse orientation. The active site Lys 201 is within hydrogen bonding distance of the carboxyl oxygen of the analog. Loop 6 (residues 330-339) remains open and flexible upon binding of the analog in the unactivated enzyme, in contrast to the closed and ordered loop 6 in the activated enzyme complex. The transition state analog is exposed to solvent due to the open conformation of loop 6. PMID:8142899

Closure Report for Corrective Action Unit 117: Area 26 Pluto Disassembly Facility, Nevada Test Site, Nevada, Revision 0

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mark Burmeister

This Closure Report (CR) presents information supporting the closure of Corrective Action Unit (CAU) 117: Area 26 Pluto Disassembly Facility, Nevada Test Site, Nevada. This CR complies with the requirements of the Federal Facility Agreement and Consent Order that was agreed to by the State of Nevada; U.S. Department of Energy (DOE), Environmental Management; U.S. Department of Defense; and DOE, Legacy Management. Corrective Action Unit 117 comprises Corrective Action Site (CAS) 26-41-01, Pluto Disassembly Facility, located in Area 26 of the Nevada Test Site. The purpose of this CR is to provide documentation supporting the completed corrective actions and providemore » data confirming that the closure objectives for CAU 117 were met. To achieve this, the following actions were performed: • Review the current site conditions, including the concentration and extent of contamination. • Implement any corrective actions necessary to protect human health and the environment. • Properly dispose of corrective action and investigation wastes. • Document Notice of Completion and closure of CAU 117 issued by the Nevada Division of Environmental Protection. From May 2008 through February 2009, closure activities were performed as set forth in the Streamlined Approach for Environmental Restoration Plan for Corrective Action Unit 117, Area 26 Pluto Disassembly Facility, Nevada Test Site, Nevada. The purpose of the activities as defined during the data quality objectives process were: • Determine whether contaminants of concern (COCs) are present. • If COCs are present, determine their nature and extent, implement appropriate corrective actions, and properly dispose of wastes. Analytes detected during the closure activities were evaluated against final action levels to determine COCs for CAU 117. Assessment of the data generated from closure activities indicated that the final action levels were exceeded for polychlorinated biphenyls (PCBs) reported as total Aroclor and radium-226. A corrective action was implemented to remove approximately 50 cubic yards of PCB-contaminated soil, approximately 1 cubic foot of radium-226 contaminated soil (and scabbled asphalt), and a high-efficiency particulate air filter that was determined to meet the criteria of a potential source material (PSM). Electrical and lighting components (i.e., PCB-containing ballasts and capacitors) and other materials (e.g., mercury-containing thermostats and switches, lead plugs and bricks) assumed to be PSM were also removed from Building 2201, as practical, without the need for sampling. Because the COC contamination and PSMs have been removed, clean closure of CAS 26-41-01 is recommended, and no use restrictions are required to be placed on this CAU. No further action is necessary because no other contaminants of potential concern were found above preliminary action levels. The physical end state for Building 2201 is expected to be eventual demolition to slab. The DOE, National Nuclear Security Administration Nevada Site Office provides the following recommendations: • Clean closure is the recommended corrective action for CAS 26-41-01 in CAU 117. • A Notice of Completion to the DOE, National Nuclear Security Administration Nevada Site Office is requested from the Nevada Division of Environmental Protection for closure of CAU 117. • Corrective Action Unit 117 should be moved from Appendix III to Appendix IV of the Federal Facility Agreement and Consent Order.« less

[Incidence of anaplastic tumor in structure of other histologic forms of the thyroid gland cancer].

PubMed

Vinnik, Iu A; Gorbenko, V N; Vas'ko, A R; Kikhtenko, E V; Gargin, V V

2014-01-01

The degrees of invasiveness, proliferative activity, morphofunctional activity of nuclei in the thyroidal gland tumors were studied, while analyzing material, obtained in 1343 patients, suffering thyroidal gland cancer (THGC) and operated on in 2000-2013 yrs. Morphological point quantity of malignancy (as a criterion of the tumor progression grade) and mitotic activity in cellular population were determined in various kinds of THGC. Undifferentiated (anaplastic carcinoma) type of THGC is the most malignant one. There were determined a spindle-like, giant-cell and squamous-cell forms of undifferentiated THGC. The presence of sites of differentiated cancer in 33% of histological preparations witnesses the interrelationship with the earlier existed pathological process.

Hands-On Hydrology

ERIC Educational Resources Information Center

Mathews, Catherine E.; Monroe, Louise Nelson

2004-01-01

A professional school and university collaboration enables elementary students and their teachers to explore hydrology concepts and realize the beneficial functions of wetlands. Hands-on experiences involve young students in determining water quality at field sites after laying the groundwork with activities related to the hydrologic cycle,…

Rapid urease test (RUT) for evaluation of urease activity in oral bacteria in vitro and in supragingival dental plaque ex vivo.

PubMed

Dahlén, Gunnar; Hassan, Haidar; Blomqvist, Susanne; Carlén, Anette

2018-05-18

Urease is an enzyme produced by plaque bacteria hydrolysing urea from saliva and gingival exudate into ammonia in order to regulate the pH in the dental biofilm. The aim of this study was to assess the urease activity among oral bacterial species by using the rapid urease test (RUT) in a micro-plate format and to examine whether this test could be used for measuring the urease activity in site-specific supragingival dental plaque samples ex vivo. The RUT test is based on 2% urea in peptone broth solution and with phenol red at pH 6.0. Oral bacterial species were tested for their urease activity using 100 μl of RUT test solution in the well of a micro-plate to which a 1 μl amount of cells collected after growth on blood agar plates or in broth, were added. The color change was determined after 15, 30 min, and 1 and 2 h. The reaction was graded in a 4-graded scale (none, weak, medium, strong). Ex vivo evaluation of dental plaque urease activity was tested in supragingival 1 μl plaque samples collected from 4 interproximal sites of front teeth and molars in 18 adult volunteers. The color reaction was read after 1 h in room temperature and scored as in the in vitro test. The strongest activity was registered for Staphylococcus epidermidis, Helicobacter pylori, Campylobacter ureolyticus and some strains of Haemophilus parainfluenzae, while known ureolytic species such as Streptococcus salivarius and Actinomyces naeslundii showed a weaker, variable and strain-dependent activity. Temperature had minor influence on the RUT reaction. The interproximal supragingival dental plaque between the lower central incisors (site 31/41) showed significantly higher scores compared to between the upper central incisors (site 11/21), between the upper left first molar and second premolar (site 26/25) and between the lower right second premolar and molar (site 45/46). The rapid urease test (RUT) in a micro-plate format can be used as a simple and rapid method to test urease activity in bacterial strains in vitro and as a chair-side method for testing urease activity in site-specific supragingival plaque samples ex vivo.

A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Dashuang; Li, Yongdong; Cabrera-Luque, Juan

2012-05-24

Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 {angstrom} resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS aremore » tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26{sup o} is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.« less

Structure and catalytic mechanism of LigI: insight into the amidohydrolase enzymes of cog3618 and lignin degradation.

PubMed

Hobbs, Merlin Eric; Malashkevich, Vladimir; Williams, Howard J; Xu, Chengfu; Sauder, J Michael; Burley, Stephen K; Almo, Steven C; Raushel, Frank M

2012-04-24

LigI from Sphingomonas paucimobilis catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) to 4-oxalomesaconate and 4-carboxy-2-hydroxymuconate in the degradation of lignin. This protein is a member of the amidohydrolase superfamily of enzymes. The protein was expressed in Escherichia coli and then purified to homogeneity. The purified recombinant enzyme does not contain bound metal ions, and the addition of metal chelators or divalent metal ions to the assay mixtures does not affect the rate of product formation. This is the first enzyme from the amidohydrolase superfamily that does not require a divalent metal ion for catalytic activity. The kinetic constants for the hydrolysis of PDC are 340 s(-1) and 9.8 × 10(6) M(-1) s(-1) (k(cat) and k(cat)/K(m), respectively). The pH dependence on the kinetic constants suggests that a single active site residue must be deprotonated for the hydrolysis of PDC. The site of nucleophilic attack was determined by conducting the hydrolysis of PDC in (18)O-labeled water and subsequent (13)C nuclear magnetic resonance analysis. The crystal structures of wild-type LigI and the D248A mutant in the presence of the reaction product were determined to a resolution of 1.9 Å. The C-8 and C-11 carboxylate groups of PDC are coordinated within the active site via ion pair interactions with Arg-130 and Arg-124, respectively. The hydrolytic water molecule is activated by the transfer of a proton to Asp-248. The carbonyl group of the lactone substrate is activated by electrostatic interactions with His-180, His-31, and His-33.

Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics.

PubMed

Arango Gutierrez, Erik; Mundhada, Hemanshu; Meier, Thomas; Duefel, Hartmut; Bocola, Marco; Schwaneberg, Ulrich

2013-12-15

Glucose oxidase is an oxidoreductase exhibiting a high β-D-glucose specificity and high stability which renders glucose oxidase well-suited for applications in diabetes care. Nevertheless, GOx activity is highly oxygen dependent which can lead to inaccuracies in amperometric β-D-glucose determinations. Therefore a directed evolution campaign with two rounds of random mutagenesis (SeSaM followed by epPCR), site saturation mutagenesis studies on individual positions, and one simultaneous site saturation library (OmniChange; 4 positions) was performed. A diabetes care well suited mediator (quinone diimine) was selected and the GOx variant (T30V I94V) served as starting point. For directed GOx evolution a microtiter plate detection system based on the quinone diimine mediator was developed and the well-known ABTS-assay was applied in microtiter plate format to validate oxygen independency of improved GOx variants. Two iterative rounds of random diversity generation and screening yielded to two subsets of amino acid positions which mainly improved activity (A173, A332) and oxygen independency (F414, V560). Simultaneous site saturation of all four positions with a reduced subset of amino acids using the OmniChange method yielded finally variant V7 with a 37-fold decreased oxygen dependency (mediator activity: 7.4 U/mg WT, 47.5 U/mg V7; oxygen activity: 172.3 U/mg WT, 30.1 U/mg V7). V7 is still highly β-D-glucose specific, highly active with the quinone diimine mediator and thermal resistance is retained (prerequisite for GOx coating of diabetes test stripes). The latter properties and V7's oxygen insensitivity make V7 a very promising candidate to replace standard GOx in diabetes care applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Landslide hazard assessment of the Black sea coastline (Caucasus, Russia) via drones

NASA Astrophysics Data System (ADS)

Kazeev, Andrey; Postoev, German; Fedotova, Ksenia

2017-04-01

Landslide hazard assessment of slopes of Sochi was performed along the railway between the cities Tuapse and Adler (total length 103 km). The railway passes through the territory with active development of hazardous geological processes such as landslides, rock falls and debris-flows. By the beginning of 2016, 36 landslide sites were discovered along the railway (total length 34 km), 48 rock-fall sites (length 31 km), and 5 debris-flow sites (length 0.14 km). In recent years the intensification of deformations was observed. For instance, during previous 10 years (1996¬¬-2005) 28 sudden deformations occurred due to slope processes, which caused interruptions in traffic. And in the present decade (2006-2015), 72 deformations were recorded. High landslide activity and economic loss determined the necessity of complex investigations of engineering geological conditions of landslides development and causes of its intensification. The protection strategy development was needed to minimize negative consequences. Thus, the investigations of landslide situation along the railway "Tuapse - Adler" included the categorization of landslide sites by level of hazard, with risk assessment based on numerical criteria. Preliminary evaluation of landslide hazard for the railway was conducted via the analysis of archived engineering-geological documents. 13 of 36 landslide sites (total length 13 km) were selected, reflecting the variety and peculiarities of landslide displacements on slopes (both active and inactive sites). Visual field observations of landslide slopes using drone "DJI Phantom 4" were completed during the second stage of this investigation. High-resolution photographs of landslide cirques, cracks, scarp walls, vegetation features were obtained via drone, which would have been impossible to obtain from the ground in conditions of dense subtropical vegetation cover. Possible approaches to the landslide activity and hazard assessment were evaluated: slope stability analysis, geophysical monitoring methods, analysis of critical deformations and critical velocities of displacement, the analysis of changes of conditions of landslide development during its displacement, as well as scoring approaches to landslide hazard and risk assessment. As the result, the method of probabilistic estimation of landslide activity and hazard has been proposed, based on selection and analysis of main factors, influencing landslide displacements. Slope steepness, landslide thickness, slope length, bedrock dip, slope relief, cracks, vegetation patterns and other factors were used for assessment of activity of landslide sites. The investigation was based on the proposed probabilistic method of assessment of landslide activity and hazard. The considered landslide sites were ranked by the rate of activity as inactive, potentially active and active. The most active sites were used to identify potentially the most hazardous sites. Furthermore, the following factors were additionally considered: the damage of railroad facilities due to landslide, landslide activity, thickness of landslide at the toe of the slope, bedrock stratification, the conditions for the cirque development, the position of the sliding surface relatively to the railway, the involvement of bedrock into displaced mass. As the result, the investigated railroad sites were divided into three categories: non-hazardous, potentially hazardous and hazardous. The research was supported by Russian Scientific Foundation (Project № 16-17-00125).

Immersion freezing of supermicron mineral dust particles: freezing results, testing different schemes for describing ice nucleation, and ice nucleation active site densities.

PubMed

Wheeler, M J; Mason, R H; Steunenberg, K; Wagstaff, M; Chou, C; Bertram, A K

2015-05-14

Ice nucleation on mineral dust particles is known to be an important process in the atmosphere. To accurately implement ice nucleation on mineral dust particles in atmospheric simulations, a suitable theory or scheme is desirable to describe laboratory freezing data in atmospheric models. In the following, we investigated ice nucleation by supermicron mineral dust particles [kaolinite and Arizona Test Dust (ATD)] in the immersion mode. The median freezing temperature for ATD was measured to be approximately -30 °C compared with approximately -36 °C for kaolinite. The freezing results were then used to test four different schemes previously used to describe ice nucleation in atmospheric models. In terms of ability to fit the data (quantified by calculating the reduced chi-squared values), the following order was found for ATD (from best to worst): active site, pdf-α, deterministic, single-α. For kaolinite, the following order was found (from best to worst): active site, deterministic, pdf-α, single-α. The variation in the predicted median freezing temperature per decade change in the cooling rate for each of the schemes was also compared with experimental results from other studies. The deterministic model predicts the median freezing temperature to be independent of cooling rate, while experimental results show a weak dependence on cooling rate. The single-α, pdf-α, and active site schemes all agree with the experimental results within roughly a factor of 2. On the basis of our results and previous results where different schemes were tested, the active site scheme is recommended for describing the freezing of ATD and kaolinite particles. We also used our ice nucleation results to determine the ice nucleation active site (INAS) density for the supermicron dust particles tested. Using the data, we show that the INAS densities of supermicron kaolinite and ATD particles studied here are smaller than the INAS densities of submicron kaolinite and ATD particles previously reported in the literature.

Recurrent Scapular Metastasis From Hepatoblastoma Shown on FDG PET/CT and F-DOPA PET/CT.

PubMed

Zhang, Bing; He, Qiao; Shi, Xinchong; Wang, Xiaoyan; Zhang, Xiangsong

2017-10-01

We report the case of a 4-year-old girl with a biochemical relapse (plasma α-fetoprotein of 57,987.6 μg/L) after hepatoblastoma and extrahepatic metastases removal and adjuvant chemotherapy. Abdominal ultrasound, CT, and MRI failed to determine the site of recurrence. F-FDG PET/CT showed increased activity in the region of left scapula and adjacent soft tissue, which was incorrectly interpreted as the postoperative repair or inflammatory change. F-DOPA PET/CT showed increased activity and noticeable progressed lesion in the same place. Finally, the left scapula was identified as the site of recurrent metastasis from hepatoblastoma by pathological examination.

Data from a thick unsaturated zone underlying Oro Grande and Sheep Creek washes in the western part of the Mojave Desert, near Victorville, San Bernardino County, California

USGS Publications Warehouse

Izbicki, John A.; Clark, Dennis A.; Pimental, Maria I.; Land, Michael; Radyk, John C.; Michel, Robert L.

2000-01-01

This report presents data on the physical properties of unsaturated alluvial deposits and on the chemical and isotopic composition of soil water and soil gas collected at 12 monitoring sites in the western part of the Mojave Desert, near Victorville, California. Sites were installed using the ODEX air-hammer method. Seven sites were located in the active channels of Oro Grande and Sheep Creek Washes. The remaining five sites were located away from the active washes. Most sites were drilled to a depth of about 100 feet below land surface; two sites were drilled to the water table almost 650 feet below land surface. Drilling procedures, lithologic and geophysical data, and site construction and instrumentation are described. Core material was analyzed for water content, bulk density, water potential, particle size, and water retention. The chemical composition of leachate from almost 1,000 subsamples of cores and cuttings was determined. Water extracted from selected subsamples of cores was analyzed for tritium and the stable isotopes of oxygen and hydrogen. Water from suction-cup lysimeters and soil-gas samples also were analyzed for chemical and isotopic composition. In addition, data on the chemical and isotopic composition of bulk precipitation from five sites and on ground water from two water-table wells are reported.

Spatio-Temporal Dynamics of Field Cricket Calling Behaviour: Implications for Female Mate Search and Mate Choice.

PubMed

Nandi, Diptarup; Balakrishnan, Rohini

2016-01-01

Amount of calling activity (calling effort) is a strong determinant of male mating success in species such as orthopterans and anurans that use acoustic communication in the context of mating behaviour. While many studies in crickets have investigated the determinants of calling effort, patterns of variability in male calling effort in natural choruses remain largely unexplored. Within-individual variability in calling activity across multiple nights of calling can influence female mate search and mate choice strategies. Moreover, calling site fidelity across multiple nights of calling can also affect the female mate sampling strategy. We therefore investigated the spatio-temporal dynamics of acoustic signaling behaviour in a wild population of the field cricket species Plebeiogryllus guttiventris. We first studied the consistency of calling activity by quantifying variation in male calling effort across multiple nights of calling using repeatability analysis. Callers were inconsistent in their calling effort across nights and did not optimize nightly calling effort to increase their total number of nights spent calling. We also estimated calling site fidelity of males across multiple nights by quantifying movement of callers. Callers frequently changed their calling sites across calling nights with substantial displacement but without any significant directionality. Finally, we investigated trade-offs between within-night calling effort and energetically expensive calling song features such as call intensity and chirp rate. Calling effort was not correlated with any of the calling song features, suggesting that energetically expensive song features do not constrain male calling effort. The two key features of signaling behaviour, calling effort and call intensity, which determine the duration and spatial coverage of the sexual signal, are therefore uncorrelated and function independently.

A dopamine D2 receptor mutant capable of G protein-mediated signaling but deficient in arrestin binding.

PubMed

Lan, Hongxiang; Liu, Yong; Bell, Michal I; Gurevich, Vsevolod V; Neve, Kim A

2009-01-01

Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D(2) and D(3) receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D(2) and D(3) receptors other than phosphorylation sites and to create a D(2) receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D(2) and D(3) receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D(2) receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D(2)-IC3 and D(3)-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D(2) and D(3) receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D(2) receptor is a key element of the arrestin binding site.

Factors in adoption of a fire department wellness program: champ-and-chief model.

PubMed

Kuehl, Hannah; Mabry, Linda; Elliot, Diane L; Kuehl, Kerry S; Favorite, Kim C

2013-04-01

To identify and evaluate determinants of fire departments' wellness program adoption. The Promoting Healthy Lifestyles: Alternative Models' Effects fire service wellness program was offered for free to all medium-sized fire departments in Oregon and Washington. An invitation to participate was mailed to key fire department decision makers (chief, union president, and wellness officer). These key decision makers from 12 sites that adopted the program and 24 matched nonadopting sites were interviewed and results were analyzed to define adoption determinants. Three adoption requirements were identified: (1) mailer connection, (2) local firefighter wellness champion, and (3) willing fire chief, whereas a fourth set of organizational factors had little or no impact on adoption including previous and ongoing wellness activities, financial pressures, and resistance to change. Findings identified determinants of medium-sized fire service wellness program adoption.

An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, William J; Senkovich, Olga; Chattopadhyay, Debasish

2009-06-08

The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips tomore » the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2{angstrom} resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits. The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.« less

RADON LEVELS AND ЕQUIVALENT DOSE RATES AT THE IRT-SOFIA RESEARCH REACTOR SITE.

PubMed

Krezhov, Kiril; Mladenov, Aleksander; Dimitrov, Dobromir

2018-06-11

Results from radon measurements by active sampling of indoor air in the buildings within the Nuclear Scientific Experimental and Educational Centre (NSEEC) protected site at the Institute for Nuclear Research and Nuclear Energy (INRNE) are presented. The inspected buildings included in this report are the IRT research reactor structure and several auxiliary formations wherein the laundry facilities and the gamma irradiator GOU-1 (60Co source) are installed as well as the Central Alarm Station (CAS) premises. Besides the reactor hall and the primary cooling loop area, special attention was given to the premises of the First Class Radiochemical Laboratory in the IRT reactor basement. Determination of radon concentration distribution in the premises of the constructions within the site is an important part of radiation surveillance during the operation and maintenance of the NSEEC facilities as well as for their involvement in the educational activities at INRNE.

Two Active Site Divalent Ions in the Crystal Structure of the Hammerhead Ribozyme Bound to a Transition State Analogue.

PubMed

Mir, Aamir; Golden, Barbara L

2016-02-02

The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analogue vanadate reveals significant rearrangements relative to the previously determined structures. The active site contracts, bringing G10.1 closer to the cleavage site and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to O6 of G12. This metal ion is well-placed to help tune the pKA of G12. On the basis of this crystal structure as well as a wealth of biochemical studies, we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid.

Active Collision Avoidance for Planetary Landers

NASA Technical Reports Server (NTRS)

Rickman, Doug; Hannan, Mike; Srinivasan, Karthik

2014-01-01

Present day robotic missions to other planets require precise, a priori knowledge of the terrain to pre-determine a landing spot that is safe. Landing sites can be miles from the mission objective, or, mission objectives may be tailored to suit landing sites. Future robotic exploration missions should be capable of autonomously identifying a safe landing target within a specified target area selected by mission requirements. Such autonomous landing sites must (1) 'see' the surface, (2) identify a target, and (3) land the vehicle. Recent advances in radar technology have resulted in small, lightweight, low power radars that are used for collision avoidance and cruise control systems in automobiles. Such radar systems can be adapted for use as active hazard avoidance systems for planetary landers. The focus of this CIF proposal is to leverage earlier work on collision avoidance systems for MSFC's Mighty Eagle lander and evaluate the use of automotive radar systems for collision avoidance in planetary landers.

Expansion of Protein Farnesyltransferase Specificity Using “Tunable” Active Site Interactions

PubMed Central

Hougland, James L.; Gangopadhyay, Soumyashree A.; Fierke, Carol A.

2012-01-01

Post-translational modifications play essential roles in regulating protein structure and function. Protein farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be “tuned” using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a protein. PMID:22992747

Two active site divalent ions in the crystal structure of the hammerhead ribozyme bound to a transition state analogue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mir, Aamir; Golden, Barbara L.

2015-11-09

The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analogue vanadate reveals significant rearrangements relative to the previously determined structures. The active site contracts, bringing G10.1 closer to the cleavage site and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to O6 of G12. This metal ion is well-placed to help tune the p K A of G12. Finally, on the basis ofmore » this crystal structure as well as a wealth of biochemical studies, in this paper we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid.« less

Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair

PubMed Central

Shinohara, Takeshi; Ikawa, Shukuko; Iwasaki, Wakana; Hiraki, Toshiki; Hikima, Takaaki; Mikawa, Tsutomu; Arai, Naoto; Kamiya, Nobuo; Shibata, Takehiko

2015-01-01

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions. PMID:25561575

Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin.

PubMed

Rostagno, A A; Schwarzbauer, J E; Gold, L I

1999-03-01

Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction.

Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin.

PubMed Central

Rostagno, A A; Schwarzbauer, J E; Gold, L I

1999-01-01

Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction. PMID:10024513

Crystal structure of the cytoplasmic phosphatase and tensin homolog (PTEN)-like region of Ciona intestinalis voltage-sensing phosphatase provides insight into substrate specificity and redox regulation of the phosphoinositide phosphatase activity.

PubMed

Matsuda, Makoto; Takeshita, Kohei; Kurokawa, Tatsuki; Sakata, Souhei; Suzuki, Mamoru; Yamashita, Eiki; Okamura, Yasushi; Nakagawa, Atsushi

2011-07-01

Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) has a transmembrane voltage sensor domain and a cytoplasmic region sharing similarity to the phosphatase and tensin homolog (PTEN). It dephosphorylates phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon membrane depolarization. The cytoplasmic region is composed of a phosphatase domain and a putative membrane interaction domain, C2. Here we determined the crystal structures of the Ci-VSP cytoplasmic region in three distinct constructs, wild-type (248-576), wild-type (236-576), and G365A mutant (248-576). The crystal structure of WT-236 and G365A-248 had the disulfide bond between the catalytic residue Cys-363 and the adjacent residue Cys-310. On the other hand, the disulfide bond was not present in the crystal structure of WT-248. These suggest the possibility that Ci-VSP is regulated by reactive oxygen species as found in PTEN. These structures also revealed that the conformation of the TI loop in the active site of the Ci-VSP cytoplasmic region was distinct from the corresponding region of PTEN; Ci-VSP has glutamic acid (Glu-411) in the TI loop, orienting toward the center of active site pocket. Mutation of Glu-411 led to acquirement of increased activity toward phosphatidylinositol 3,5-bisphosphate, suggesting that this site is required for determining substrate specificity. Our results provide the basic information of the enzymatic mechanism of Ci-VSP.

The molecular architecture of human N-acetylgalactosamine kinase.

PubMed

Thoden, James B; Holden, Hazel M

2005-09-23

Galactokinase plays a key role in normal galactose metabolism by catalyzing the conversion of alpha-d-galactose to galactose 1-phosphate. Within recent years, the three-dimensional structures of human galactokinase and two bacterial forms of the enzyme have been determined. Originally, the gene encoding galactokinase in humans was mapped to chromosome 17. An additional gene, encoding a protein with sequence similarity to galactokinase, was subsequently mapped to chromosome 15. Recent reports have shown that this second gene (GALK2) encodes an enzyme with greater activity against GalNAc than galactose. This enzyme, GalNAc kinase, has been implicated in a salvage pathway for the reutilization of free GalNAc derived from the degradation of complex carbohydrates. Here we report the first structural analysis of a GalNAc kinase. The structure of the human enzyme was solved in the presence of MnAMPPNP and GalNAc or MgATP and GalNAc (which resulted in bound products in the active site). The enzyme displays a distinctly bilobal appearance with its active site wedged between the two domains. The N-terminal region is dominated by a seven-stranded mixed beta-sheet, whereas the C-terminal motif contains two layers of anti-parallel beta-sheet. The overall topology displayed by GalNAc kinase places it into the GHMP superfamily of enzymes, which generally function as small molecule kinases. From this investigation, the geometry of the GalNAc kinase active site before and after catalysis has been revealed, and the determinants of substrate specificity have been defined on a molecular level.

Effect of Methodological and Ecological Approaches on Heterogeneity of Nest-Site Selection of a Long-Lived Vulture

PubMed Central

Moreno-Opo, Rubén; Fernández-Olalla, Mariana; Margalida, Antoni; Arredondo, Ángel; Guil, Francisco

2012-01-01

The application of scientific-based conservation measures requires that sampling methodologies in studies modelling similar ecological aspects produce comparable results making easier their interpretation. We aimed to show how the choice of different methodological and ecological approaches can affect conclusions in nest-site selection studies along different Palearctic meta-populations of an indicator species. First, a multivariate analysis of the variables affecting nest-site selection in a breeding colony of cinereous vulture (Aegypius monachus) in central Spain was performed. Then, a meta-analysis was applied to establish how methodological and habitat-type factors determine differences and similarities in the results obtained by previous studies that have modelled the forest breeding habitat of the species. Our results revealed patterns in nesting-habitat modelling by the cinereous vulture throughout its whole range: steep and south-facing slopes, great cover of large trees and distance to human activities were generally selected. The ratio and situation of the studied plots (nests/random), the use of plots vs. polygons as sampling units and the number of years of data set determined the variability explained by the model. Moreover, a greater size of the breeding colony implied that ecological and geomorphological variables at landscape level were more influential. Additionally, human activities affected in greater proportion to colonies situated in Mediterranean forests. For the first time, a meta-analysis regarding the factors determining nest-site selection heterogeneity for a single species at broad scale was achieved. It is essential to homogenize and coordinate experimental design in modelling the selection of species' ecological requirements in order to avoid that differences in results among studies would be due to methodological heterogeneity. This would optimize best conservation and management practices for habitats and species in a global context. PMID:22413023

Seasonal and spatial variation in reactive oxygen species activity of quasi-ultrafine particles (PM0.25) in the Los Angeles metropolitan area and its association with chemical composition

NASA Astrophysics Data System (ADS)

Saffari, Arian; Daher, Nancy; Shafer, Martin M.; Schauer, James J.; Sioutas, Constantinos

2013-11-01

Seasonal and spatial variation in redox activity of quasi-ultrafine particles (PM0.25) and its association with chemical species was investigated at 9 distinct sampling sites across the Los Angeles metropolitan area. Biologically reactive oxygen species (ROS) assay (generation of ROS in rat alveolar macrophage cells) was employed in order to assess the redox activity of PM0.25 samples. Seasonally, fall and summer displayed higher volume-based ROS activity (i.e. ROS activity per unit volume of air) compared to spring and winter. ROS levels were generally higher at near source and urban background sites compared to rural receptor locations, except for summer when comparable ROS activity was observed at the rural receptor sites. Univariate linear regression analysis indicated association (R > 0.7) between ROS activity and organic carbon (OC), water soluble organic carbon (WSOC) and water soluble transition metals (including Fe, V, Cr, Cd, Ni, Zn, Mn, Pb and Cu). A multivariate regression method was also used to obtain a model to predict the ROS activity of PM0.25, based on its water-soluble components. The most important species associated with ROS were Cu and La at the source site of Long Beach, and Fe and V at urban Los Angeles sites. These metals are tracers of road dust enriched with vehicular emissions (Fe and Cu) and residual oil combustion (V and La). At Riverside, a rural receptor location, WSOC and Ni (tracers of secondary organic aerosol and metal plating, respectively) were the dominant species driving the ROS activity. At Long Beach, the multivariate model was able to reconstruct the ROS activity with a high coefficient of determination (R2 = 0.82). For Los Angeles and Riverside, however, the regression models could only explain 63% and 68% of the ROS activity, respectively. The unexplained portion of the measured ROS activity is likely attributed to the nature of organic species not captured in the organic carbon (OC) measurement as well as non-linear effects, which were not included in our linear model.

A Methodology to Evaluate Ecological Resources and Risk Using Two Case Studies at the Department of Energy’s Hanford Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burger, Joanna; Gochfeld, Michael; Bunn, Amoret

An assessment of the potential risks to ecological resources from remediation activities or other perturbations should involve a quantitative evaluation of resources on the remediation site and in the surrounding environment. We developed a risk methodology to rapidly evaluate potential impact on ecological resources for the U.S. Department of Energy’s Hanford Site in southcentral Washington State. We describe the application of the risk evaluation for two case studies to illustrate its applicability. The ecological assessment involves examining previous sources of information for the site, defining different resource levels from 0 to 5. We also developed a risk rating scale frommore » nondiscernable to very high. Field assessment is the critical step to determine resource levels or to determine if current conditions are the same as previously evaluated. We provide a rapid assessment method for current ecological conditions that can be compared to previous site-specific data, or that can be used to assess resource value on other sites where ecological information is not generally available. The method is applicable to other Department of Energy’s sites, where its development may involve a range of state regulators, resource trustees, Tribes and other stakeholders. Achieving consistency across Department of Energy’s sites for valuation of ecological resources on remediation sites will assure Congress and the public that funds and personnel are being deployed appropriately.« less

Effects of Alien Plants on Ecosystem Structure and Functioning and Implications for Restoration: Insights from Three Degraded Sites in South African Fynbos

NASA Astrophysics Data System (ADS)

Gaertner, Mirijam; Richardson, David M.; Privett, Sean D. J.

2011-07-01

We investigated the type and extent of degradation at three sites on the Agulhas Plain, South Africa: an old field dominated by the alien grass Pennisetum clandestinum Pers . (kikuyu), an abandoned Eucalyptus plantation, and a natural fynbos community invaded by nitrogen fixing—Australian Acacia species. These forms of degradation are representative of many areas in the region. By identifying the nature and degree of ecosystem degradation we aimed to determine appropriate strategies for restoration in this biodiversity hotspot. Vegetation surveys were conducted at degraded sites and carefully selected reference sites. Soil-stored propagule seed banks and macro- and micro-soil nutrients were determined. Species richness, diversity and native cover under Eucalyptus were extremely low compared to the reference site and alterations of the soil nutrients were the most severe. The cover of indigenous species under Acacia did not differ significantly from that in reference sites, but species richness was lower under Acacia and soils were considerably enriched. Native species richness was much lower in the kikuyu site, but soil nutrient status was similar to the reference site. Removal of the alien species alone may be sufficient to re-initiate ecosystem recovery at the kikuyu site, whereas active restoration is required to restore functioning ecosystems dominated by native species in the Acacia thicket and the Eucalyptus plantation. To restore native plant communities we suggest burning, mulching with sawdust and sowing of native species.

Productivity, pesticides, and management of the Peregrine Falcon in Arizona

USGS Publications Warehouse

Ellis, D.H.

1985-01-01

In the decade since research commenced with the Peregrine in Arizona, over 60 sites have been identified which historically or presently are occupied by breeding pairs. Productivity was determined for about 120 breeding attempts from 1975-85. Almost all sites, for which productivity information is available for two or more years, have hatched young. Average values for fledging success were ca. 1.4 young/attempt for all active sites and ca. 2.3 young/attempt for successful sites. Eggshell thickness values were highly varied, but few samples reflect thinning sufficient to cause reproductive failure, and the population appears to be increasing slightly. Management practices which can further benefit the falcon include: controlling pesticide use, habitat protection, and information management.

Biochemical changes in longear sunfish, Lepomis megalotis, associated with lead, cadmium and zinc from mine tailings

USGS Publications Warehouse

Dwyer, F.J.; Schmitt, C.J.; Finger, S.E.; Mehrle, P.M.

1988-01-01

Longear sunfish were collected from a stream contaminated with mine tailings rich in lead (Pb), cadmium (Cd) and zinc (Zn). Blood samples were analysed for δ-aminolevulinic acid dehydratase (ALA-D) activity and Pb concentration. Vertebrae were tested for bone strength and composition, and Pb, Zn, and Cd concentrations were determined in muscle tissue. ALA-D activity was negatively correlated with blood Pb concentration (r=–0.66), and enzyme activity was significantly higher and blood Pb significantly lower at the reference site than at the contaminated sites. Blood Pb was highly correlated with Pb in muscle tissue (r= 0.72), and the concentrations of Pb and Cd in muscle tissues were themselves correlated (r= 0.64). In fish from contaminated sites, two of the mechanical properties of the vertebrae measured (elastic limit and modulus of elasticity) were significantly different from values in fish from the reference site. These properties and one other (stress) were weakly correlated with muscle Cd concentration (0.42 < r < 0.46). Biochemical differences among fish from different sites were also evident; concentrations of calcium, phosphorus and collagen were lower in the vertebrae of fish from some of the contaminated sites than at the reference site, and bone phosphorus was negatively correlated with concentrations of Pb in both muscle (r=– 0.62) and blood (r=– 0.75). Collectively, these results indicate that, in addition to the well-documented effects of Pb on haem synthesis, other important biochemical pathways may be disrupted by continuous low-level exposure to elemental contaminants.

Chromosomal damage and EROD induction in tree swallows (Tachycineta bicolor) along the Upper Mississippi River, Minnesota, USA

USGS Publications Warehouse

Emilie Bigorgne,; Custer, Thomas W.; Dummer, Paul; Erickson, Richard A.; Karouna-Renier, Natalie K.; Schultz, Sandra; Custer, Christine M.; Thogmartin, Wayne E.; Cole W. Matson,

2015-01-01

The health of tree swallows, Tachycineta bicolor, on the Upper Mississippi River (UMR) was assessed in 2010 and 2011 using biomarkers at six sites downriver of Minneapolis/St. Paul, MN metropolitan area, a tributary into the UMR, and a nearby lake. Chromosomal damage was evaluated in nestling blood by measuring the coefficient of variation of DNA content (DNA CV) using flow cytometry. Cytochrome P450 1A activity in nestling liver was measured using the ethoxyresorufin-O-dealkylase (EROD) assay, and oxidative stress was estimated in nestling livers via determination of thiobarbituric acid reacting substances (TBARS), reduced glutathione (GSH), oxidized glutathione (GSSG), the ratio GSSG/GSH, total sulfhydryl, and protein bound sulfhydryl (PBSH). A multilevel regression model (DNA CV) and simple regressions (EROD and oxidative stress) were used to evaluate biomarker responses for each location. Chromosomal damage was significantly elevated at two sites on the UMR (Pigs Eye and Pool 2) relative to the Green Mountain Lake reference site, while the induction of EROD activity was only observed at Pigs Eye. No measures of oxidative stress differed among sites. Multivariate analysis confirmed an increased DNA CV at Pigs Eye and Pool 2, and elevated EROD activity at Pigs Eye. These results suggest that the health of tree swallows has been altered at the DNA level at Pigs Eye and Pool 2 sites, and at the physiological level at Pigs Eye site only.

Residual herbicide study on selected Hanford Site roadsides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, J.L.; Kemp, C.J.; Sackschewsky, M.R.

Westinghouse Hanford Company routinely treats roadsides with herbicides to control undesirable plant growth. An experiment was conducted to test perennial grass germination in soils adjacent to roadways of the Hanford Site. The primary variable was the distance from the roadside. A simple germination test was executed in a controlled-environment chamber to determine the residual effects of these applications. As expected, the greatest herbicide activity was found directly adjacent to the roadway, approximately 0 to 20 ft (0 to 6.3 m) from the roadway.

Decreased glutathione S-transferase expression and activity and altered sex steroids in Lake Apopka brown bullheads (Ameriurus nebulosus)

USGS Publications Warehouse

Gallagher, E.P.; Gross, T.S.; Sheehy, K.M.

2001-01-01

A number of freshwater lakes and reclaimed agricultural sites in Central Florida have been the receiving waters for agrochemical and municipal runoff. One of these sites, Lake Apopka, is also a eutrophic system that has been the focus of several case studies reporting altered reproductive activity linked to bioaccumulation of persistent organochlorine chemicals in aquatic species. The present study was initiated to determine if brown bullheads (Ameriurus nebulosus) from the north marsh of Lake Apopka (Lake Apopka Marsh) exhibit an altered capacity to detoxify environmental chemicals through hepatic glutathione S-transferase (GST)-mediated conjugation as compared with bullheads from a nearby reference site (Lake Woodruff). We also compared plasma sex hormone concentrations (testosterone, 17-?? estradiol, and 11 keto-testosterone) in bullheads from the two sites. Female bullheads from Lake Apopka had 40% lower initial rate GST conjugative activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 50% lower activity towards p-nitrobutyl chloride (NBC), 33% lower activity toward ethacrynic acid (ECA), and 43% lower activity toward ??5-androstene-3,17-dione (??5-ADI), as compared with female bullheads from Lake Woodruff. Enzyme kinetic analyses demonstrated that female bullheads from Lake Apopka had lower GST-catalyzed CDNB clearance than did female Lake Woodruff bullheads. Western blotting studies of bullhead liver cytosolic proteins demonstrated that the reduced GST catalytic activities in female Lake Apopka bullheads were accompanied by lower expression of hepatic GST protein. No site differences were observed with respect to GST activities or GST protein expression in male bullheads. Female Lake Apopka bullheads also had elevated concentrations of plasma androgens (testosterone and 11-ketotestosterone) as compared with females from Lake Woodruff. In contrast, male Lake Apopka bullheads had elevated levels of plasma estrogen but similar levels of androgens as compared with male bullheads from Lake Woodruff. Collectively, our studies indicate the presence of reduced GST protein expression, reduced GST conjugative capacity and altered sex steroid homeostasis in female bullheads from a contaminated field site in Central Florida. The implications of these physiological alterations in terms of pollutant biotransformation and reproduction are discussed. ?? 2001 Elsevier Science B.V. All rights reserved.

The Mode of Inhibitor Binding to Peptidyl-tRNA Hydrolase: Binding Studies and Structure Determination of Unbound and Bound Peptidyl-tRNA Hydrolase from Acinetobacter baumannii

PubMed Central

Kaushik, Sanket; Singh, Nagendra; Yamini, Shavait; Singh, Avinash; Sinha, Mau; Arora, Ashish; Kaur, Punit; Sharma, Sujata; Singh, Tej P.

2013-01-01

The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design. PMID:23844024

Adsorption Thermodynamics and Intrinsic Activation Parameters for Monomolecular Cracking of n -Alkanes on Brønsted Acid Sites in Zeolites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Janda, Amber; Vlaisavljevich, Bess; Lin, Li-Chiang

Experimental measurements of the rate coefficient (kapp) and apparent enthalpies and entropies of activation (ΔHapp and ΔSapp) for alkane cracking catalyzed by acidic zeolites can be used to characterize the effects of zeolite structure and alkane size on the intrinsic enthalpy and entropy of activation, ΔHint‡ and ΔSint‡. To determine ΔHint‡ and ΔSint‡, enthalpies and entropies of adsorption, ΔHads-H+ and ΔSads-H+, must be determined for alkane molecules moving from the gas phase to Brønsted acid sites at reaction temperatures (>673 K). Experimental values of ΔHapp and ΔSapp must also be properly defined in terms of ΔHads-H+ and ΔSads-H+. We reportmore » here a method for determining ΔHads-H+ and ΔSads-H+ in which the adsorption site is represented by a fixed volume that includes the proton. Values of ΔHads-H+ and ΔSads-H+ obtained from Monte Carlo simulations are in good agreement with values obtained from experimental data measured at 300–400 K. An important feature of the simulations, however, is their ability to account for the redistribution of alkane adsorbed at protons in different locations with increasing temperature. Values of ΔHint‡ and ΔSint‡ for the cracking of propane through n-hexane, determined from measured values of kapp and ΔHapp and simulated values of ΔHads-H+ and ΔSads-H+, agree well with values obtained independently from quantum mechanics/molecular mechanics calculations. Application of our method of analysis reveals that the observed increase in kapp with increasing n-alkane size is due primarily to a decrease in ΔHint‡ with increasing chain length and that ΔSint‡ is independent of chain length.« less

S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK) channels

PubMed Central

Shipston, Michael J.

2014-01-01

Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation) represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs) and acyl thioesterases (APTs). S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signaling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease. PMID:25140154

Water-Quality, Bed-Sediment, and Biological Data (October 2007 through September 2008) and Statistical Summaries of Long-Term Data for Streams in the Clark Fork Basin, Montana

USGS Publications Warehouse

Dodge, Kent A.; Hornberger, Michelle I.; Dyke, Jessica

2009-01-01

Water, bed sediment, and biota were sampled in streams from Butte to near Missoula as part of a long-term monitoring program in the upper Clark Fork basin; additional water samples were collected in the Clark Fork basin from sites near Missoula downstream to near the confluence of the Clark Fork and Flathead River as part of a supplemental sampling program. The sampling programs were conducted in cooperation with the U.S. Environmental Protection Agency to characterize aquatic resources in the Clark Fork basin of western Montana, with emphasis on trace elements associated with historic mining and smelting activities. Sampling sites were located on the Clark Fork and selected tributaries. Water samples were collected periodically at 23 sites from October 2007 through September 2008. Bed-sediment and biota samples were collected once at 13 sites during August 2008. This report presents the analytical results and quality assurance data for water-quality, bed-sediment, and biota samples collected at all long-term and supplemental monitoring sites from October 2007 through September 2008. Water-quality data include concentrations of selected major ions, trace elements, and suspended sediment. Turbidity was analyzed for water samples collected at sites where seasonal daily values of turbidity were being determined and at Clark Fork above Missoula. Nutrients also were analyzed at all the supplemental water-quality sites, except for Clark Fork Bypass, near Bonner. Daily values of suspended-sediment concentration and suspended-sediment discharge were determined for four sites, and seasonal daily values of turbidity were determined for four sites. Bed-sediment data include trace-element concentrations in the fine-grained fraction. Biological data include trace-element concentrations in whole-body tissue of aquatic benthic insects. Statistical summaries of long-term water-quality, bed-sediment, and biological data for sites in the upper Clark Fork basin are provided for the period of record since 1985.

Non-Invasive Seismic Methods for Earthquake Site Classification Applied to Ontario Bridge Sites

NASA Astrophysics Data System (ADS)

Bilson Darko, A.; Molnar, S.; Sadrekarimi, A.

2017-12-01

How a site responds to earthquake shaking and its corresponding damage is largely influenced by the underlying ground conditions through which it propagates. The effects of site conditions on propagating seismic waves can be predicted from measurements of the shear wave velocity (Vs) of the soil layer(s) and the impedance ratio between bedrock and soil. Currently the seismic design of new buildings and bridges (2015 Canadian building and bridge codes) requires determination of the time-averaged shear-wave velocity of the upper 30 metres (Vs30) of a given site. In this study, two in situ Vs profiling methods; Multichannel Analysis of Surface Waves (MASW) and Ambient Vibration Array (AVA) methods are used to determine Vs30 at chosen bridge sites in Ontario, Canada. Both active-source (MASW) and passive-source (AVA) surface wave methods are used at each bridge site to obtain Rayleigh-wave phase velocities over a wide frequency bandwidth. The dispersion curve is jointly inverted with each site's amplification function (microtremor horizontal-to-vertical spectral ratio) to obtain shear-wave velocity profile(s). We apply our non-invasive testing at three major infrastructure projects, e.g., five bridge sites along the Rt. Hon. Herb Gray Parkway in Windsor, Ontario. Our non-invasive testing is co-located with previous invasive testing, including Standard Penetration Test (SPT), Cone Penetration Test and downhole Vs data. Correlations between SPT blowcount and Vs are developed for the different soil types sampled at our Ontario bridge sites. A robust earthquake site classification procedure (reliable Vs30 estimates) for bridge sites across Ontario is evaluated from available combinations of invasive and non-invasive site characterization methods.

Fine-tuning the onset of myogenesis by homeobox proteins that interact with the Myf5 limb enhancer

PubMed Central

Daubas, Philippe; Duval, Nathalie; Bajard, Lola; Langa Vives, Francina; Robert, Benoît; Mankoo, Baljinder S.; Buckingham, Margaret

2015-01-01

ABSTRACT Skeletal myogenesis in vertebrates is initiated at different sites of skeletal muscle formation during development, by activation of specific control elements of the myogenic regulatory genes. In the mouse embryo, Myf5 is the first myogenic determination gene to be expressed and its spatiotemporal regulation requires multiple enhancer sequences, extending over 120 kb upstream of the Mrf4-Myf5 locus. An enhancer, located at −57/−58 kb from Myf5, is responsible for its activation in myogenic cells derived from the hypaxial domain of the somite, that will form limb muscles. Pax3 and Six1/4 transcription factors are essential activators of this enhancer, acting on a 145-bp core element. Myogenic progenitor cells that will form the future muscle masses of the limbs express the factors necessary for Myf5 activation when they delaminate from the hypaxial dermomyotome and migrate into the forelimb bud, however they do not activate Myf5 and the myogenic programme until they have populated the prospective muscle masses. We show that Msx1 and Meox2 homeodomain-containing transcription factors bind in vitro and in vivo to specific sites in the 145-bp element, and are implicated in fine-tuning activation of Myf5 in the forelimb. Msx1, when bound between Pax and Six sites, prevents the binding of these key activators, thus inhibiting transcription of Myf5 and consequent premature myogenic differentiation. Meox2 is required for Myf5 activation at the onset of myogenesis via direct binding to other homeodomain sites in this sequence. Thus, these homeodomain factors, acting in addition to Pax3 and Six1/4, fine-tune the entry of progenitor cells into myogenesis at early stages of forelimb development. PMID:26538636

Diurnal and Seasonal Variations of Eddy-Covariance Carbon Dioxide Fluxes Above an Urban Wetland, Partitioned by Vegetation Cover

NASA Astrophysics Data System (ADS)

Schafer, K. V.; Duman, T.

2017-12-01

The New Jersey Meadowlands are an urban brackish marsh with a long history of human activity causing disturbances and alterations. Carbon emissions were measured from two sites in the Meadowlands, a natural site and a restored site, using eddy-covariance (EC) from 2014 to 2016. At each site, the EC towers were placed at the interface of two vegetation covers, allowing capturing this aspect of the wetland's heterogeneity. Using footprint modeling and light response curves we were able to partition measured fluxes between vegetation cover types and compare CO2 fluxes from patches of invasive versus native wetland vegetation communities. We show that further separating the data into seasonal and diurnal fluxes reveals patterns in CO2 fluxes that allow determining the nature of each vegetation cover as a source or sink for CO2. Our results also show that CO2 emissions from the restored wetland are significantly higher than the natural wetland. Areas of invasive Phragmites australis at the natural site had the lowest CO2 release rates during winter. These were consistently lower in magnitude than summer daytime uptake, therefore making this part of the wetland a CO2 sink. Areas planted with native Spartina alterniflora at the restored site had the largest uptake during daytime, therefore seemingly justifying restoration activities. However, they also had the highest emission rates during summer nighttime, and therefore the daily summer net uptake was not the highest compared with other vegetation covers. Furthermore, emissions from the restored site during winter were larger compared to the natural site, indicating that restoration activities might have led to a significant increase of carbon release from the wetland. Thus, during the study period the restored wetland acted as a carbon source.

The Crystal Structure of the Ivy delta4-16:0-ACP Desaturase Reveals Structural Details of the Oxidized Active Site and Potential Determinants of Regioselectivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guy,J.; Whittle, E.; Kumaran, D.

2007-01-01

The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the {Delta}{sup 4} desaturation of 16:0-ACP and the{Delta}{sup 9} desaturation of 18:0-ACP and further desaturates{Delta}{sup 9}-16:1 or {Delta}{sup 9}-18:1 to the corresponding {Delta}{sup 4,9} dienes. The crystal structure of the enzyme has been solved to 1.95{angstrom} resolution, and both the iron-iron distance of 3.2{angstrom} and the presence of a {mu}-oxo bridge reveal this to be the only reported structure of a desaturase in the oxidized FeIII-FeIII form. Significant differences are seen between the oxidized active site and the reduced active site of the Ricinus communis (castor) desaturase;more » His{sup 227} coordination to Fe2 is lost, and the side chain of Glu{sup 224}, which bridges the two iron ions in the reduced structure, does not interact with either iron. Although carboxylate shifts have been observed on oxidation of other diiron proteins, this is the first example of the residue moving beyond the coordination range of both iron ions. Comparison of the ivy and castor structures reveal surface amino acids close to the annulus of the substrate-binding cavity and others lining the lower portion of the cavity that are potential determinants of their distinct substrate specificities. We propose a hypothesis that differences in side chain packing explains the apparent paradox that several residues lining the lower portion of the cavity in the ivy desaturase are bulkier than their equivalents in the castor enzyme despite the necessity for the ivy enzyme to accommodate three more carbons beyond the diiron site.« less

Accommodation of GDP-Linked Sugars in the Active Site of GDP-Perosamine Synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, Paul D.; Carney, Amanda E.; Holden, Hazel M.

2009-01-12

Perosamine (4-amino-4,6-dideoxy-d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue,more » which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 {angstrom} resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K{sub m} for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k{sub cat} was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy-d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.« less

SPIRAL2 Determines Plant Microtubule Organization by Modulating Microtubule Severing

PubMed Central

Wightman, Raymond; Chomicki, Guillaume; Kumar, Manoj; Carr, Paul; Turner, Simon R.

2013-01-01

Summary One of the defining characteristics of plant growth and morphology is the pivotal role of cell expansion. While the mechanical properties of the cell wall determine both the extent and direction of cell expansion, the cortical microtubule array plays a critical role in cell wall organization and, consequently, determining directional (anisotropic) cell expansion [1–6]. The microtubule-severing enzyme katanin is essential for plants to form aligned microtubule arrays [7–10]; however, increasing severing activity alone is not sufficient to drive microtubule alignment [11]. Here, we demonstrate that katanin activity depends upon the behavior of the microtubule-associated protein (MAP) SPIRAL2 (SPR2). Petiole cells in the cotyledon epidermis exhibit well-aligned microtubule arrays, whereas adjacent pavement cells exhibit unaligned arrays, even though SPR2 is found at similar levels in both cell types. In pavement cells, however, SPR2 accumulates at microtubule crossover sites, where it stabilizes these crossovers and prevents severing. In contrast, in the adjacent petiole cells, SPR2 is constantly moving along the microtubules, exposing crossover sites that become substrates for severing. Consequently, our study reveals a novel mechanism whereby microtubule organization is determined by dynamics and localization of a MAP that regulates where and when microtubule severing occurs. PMID:24055158

Glutamine 57 at the complementary binding site face is a key determinant of morantel selectivity for {alpha}7 nicotinic receptors.

PubMed

Bartos, Mariana; Price, Kerry L; Lummis, Sarah C R; Bouzat, Cecilia

2009-08-07

Nicotinic receptors (AChRs) play key roles in synaptic transmission. We explored activation of neuronal alpha7 and mammalian muscle AChRs by morantel and oxantel. Our results revealed a novel action of morantel as a high efficacy and more potent agonist than ACh of alpha7 receptors. The EC(50) for activation by morantel of both alpha7 and alpha7-5HT(3A) receptors is 7-fold lower than that determined for ACh. The minimum morantel concentration required to activate alpha7-5HT(3A) channels is 6-fold lower than that of ACh, and activation episodes are more prolonged than in the presence of ACh. By contrast, oxantel is a weak agonist of alpha7 and alpha7-5HT(3A), and both drugs are very low efficacy agonists of muscle AChRs. The replacement of Gln(57) in alpha7 by glycine, which is found in the equivalent position of the muscle AChR, decreases the efficacy for activation and turns morantel into a partial agonist. The reverse mutation in the muscle AChR (epsilonG57Q) increases 7-fold the efficacy of morantel. The mutations do not affect activation by ACh or oxantel, indicating that this position is selective for morantel. In silico studies show that the tetrahydropyrimidinyl group, common to both drugs, is close to Trp(149) of the principal face of the binding site, whereas the other cyclic group is proximal to Gln(57) of the complementary face in morantel but not in oxantel. Thus, position 57 at the complementary face is a key determinant of the high selectivity of morantel for alpha7. These results provide new information for further progress in drug design.

Atomic resolution crystal structures and quantum chemistry meet to reveal subtleties of hydroxynitrile lyase catalysis.

PubMed

Schmidt, Andrea; Gruber, Karl; Kratky, Christoph; Lamzin, Victor S

2008-08-01

Hydroxynitrile lyases are versatile enzymes that enantiospecifically cope with cyanohydrins, important intermediates in the production of various agrochemicals or pharmaceuticals. We determined four atomic resolution crystal structures of hydroxynitrile lyase from Hevea brasiliensis: one native and three complexes with acetone, isopropyl alcohol, and thiocyanate. We observed distinct distance changes among the active site residues related to proton shifts upon substrate binding. The combined use of crystallography and ab initio quantum chemical calculations allowed the determination of the protonation states in the enzyme active site. We show that His(235) of the catalytic triad must be protonated in order for catalysis to proceed, and we could reproduce the cyanohydrin synthesis in ab initio calculations. We also found evidence for the considerable pK(a) shifts that had been hypothesized earlier. We envision that this knowledge can be used to enhance the catalytic properties and the stability of the enzyme for industrial production of enantiomerically pure cyanohydrins.

Sequence signatures of allosteric proteins towards rational design.

PubMed

Namboodiri, Saritha; Verma, Chandra; Dhar, Pawan K; Giuliani, Alessandro; Nair, Achuthsankar S

2010-12-01

Allostery is the phenomenon of changes in the structure and activity of proteins that appear as a consequence of ligand binding at sites other than the active site. Studying mechanistic basis of allostery leading to protein design with predetermined functional endpoints is an important unmet need of synthetic biology. Here, we screened the amino acid sequence landscape in search of sequence-signatures of allostery using Recurrence Quantitative Analysis (RQA) method. A characteristic vector, comprised of 10 features extracted from RQA was defined for amino acid sequences. Using Principal Component Analysis, four factors were found to be important determinants of allosteric behavior. Our sequence-based predictor method shows 82.6% accuracy, 85.7% sensitivity and 77.9% specificity with the current dataset. Further, we show that Laminarity-Mean-hydrophobicity representing repeated hydrophobic patches is the most crucial indicator of allostery. To our best knowledge this is the first report that describes sequence determinants of allostery based on hydrophobicity. As an outcome of these findings, we plan to explore possibility of inducing allostery in proteins.

Mechanisms of mTORC1 activation by RHEB and inhibition by PRAS40.

PubMed

Yang, Haijuan; Jiang, Xiaolu; Li, Buren; Yang, Hyo J; Miller, Meredith; Yang, Angela; Dhar, Ankita; Pavletich, Nikola P

2017-12-21

The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and metabolism in response to nutrients, energy levels, and growth factors. It contains the atypical kinase mTOR and the RAPTOR subunit that binds to the Tor signalling sequence (TOS) motif of substrates and regulators. mTORC1 is activated by the small GTPase RHEB (Ras homologue enriched in brain) and inhibited by PRAS40. Here we present the 3.0 ångström cryo-electron microscopy structure of mTORC1 and the 3.4 ångström structure of activated RHEB-mTORC1. RHEB binds to mTOR distally from the kinase active site, yet causes a global conformational change that allosterically realigns active-site residues, accelerating catalysis. Cancer-associated hyperactivating mutations map to structural elements that maintain the inactive state, and we provide biochemical evidence that they mimic RHEB relieving auto-inhibition. We also present crystal structures of RAPTOR-TOS motif complexes that define the determinants of TOS recognition, of an mTOR FKBP12-rapamycin-binding (FRB) domain-substrate complex that establishes a second substrate-recruitment mechanism, and of a truncated mTOR-PRAS40 complex that reveals PRAS40 inhibits both substrate-recruitment sites. These findings help explain how mTORC1 selects its substrates, how its kinase activity is controlled, and how it is activated by cancer-associated mutations.

Migration of fallout radiocaesium in a grassland soil from 1986 to 2001. Part I: activity-depth profiles of (134)Cs and (137)Cs.

PubMed

Schimmack, W; Schultz, W

2006-09-15

The temporal changes of the vertical distribution of (134)Cs (deposited by the Chernobyl fallout in 1986) and (137)Cs (deposited by the Chernobyl and the global fallout) in the soil were investigated at an undisturbed Bavarian grassland site in Germany. At ten sampling dates between 1986 and 2001, the activity density of (134)Cs and (137)Cs was determined in various soil layers down to 80 cm depth. In 2001, the small-scale spatial variability of the radiocaesium activity was determined by sampling five plots within 10 m(2) (coefficient of variation about 20% for the upper soil layers). Between 1987 and 1990, substantial changes of the activity-depth profiles were observed. The percentage depth distributions of (134)Cs and (137)Cs were rather similar. The 50%-depth of the accumulated activity increased from 2.4 cm in 1988 to 5.3 cm in 2001 for (134)Cs and from 2.7 to 5.8 cm for (137)Cs. This indicates that at the study site the migration data of Chernobyl-derived (137)Cs can be estimated by those of total (137)Cs. In the second part of this study, the activity-depth profiles will be evaluated by the convection-dispersion model [Schimmack, W, Feria Márquez, F. Migration of fallout radiocaesium in a grassland soil from 1986 to 2001. Part II: Evaluation of the activity-depth profiles by transport models. Sci Total Environ 2006-this issue].

Site characterization progress report: Yucca Mountain, Nevada. Number 15, April 1--September 30, 1996

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1997-04-01

During the second half of fiscal year 1996, activities at the Yucca Mountain Site Characterization Project (Project) supported the objectives of the revised Program Plan released this period by the Office of Civilian Radioactive Waste Management of the US Department of Energy (Department). Outlined in the revised plan is a focused, integrated program of site characterization, design, engineering, environmental, and performance assessment activities that will achieve key Program and statutory objectives. The plan will result in the development of a license application for repository construction at Yucca Mountain, if the site is found suitable. Activities this period focused on twomore » of the three near-term objectives of the revised plan: updating in 1997 the regulatory framework for determining the suitability of the site for the proposed repository concept and providing information for a 1998 viability assessment of continuing toward the licensing of a repository. The Project has also developed a new design approach that uses the advanced conceptual design published during the last reporting period as a base for developing a design that will support the viability assessment. The initial construction phase of the Thermal Testing Facility was completed and the first phase of the in situ heater tests began on schedule. In addition, phase-one construction was completed for the first of two alcoves that will provide access to the Ghost Dance fault.« less

Preparation and preliminary characterization of crystallizing fluorescent derivatives of chicken egg white lysozyme

NASA Astrophysics Data System (ADS)

Sumida, John P.; Forsythe, Elizabeth L.; Pusey, Marc L.

2001-11-01

Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins are intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein molecule. Lucifer yellow and 5-(2-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS) have been attached to the side chain carboxyl of Asp 101 using a carbodiimide coupling procedure. Asp 101 lies within the active site cleft, and it is believed that the probes are "buried" within that cleft. Lucifer yellow and EDANS probes with iodoacetamide reactive groups have been bound to His 15, located on the "back side" of the molecule relative to the active site. All the derivatives fluoresce in the solution and the crystalline states. Fluorescence characterization has focused on determination of binding effects on the probe quantum yield, lifetime, absorption and emission spectra, and quenching by added solutes. Quenching studies show that, as postulated, the Asp 101-bound probes are partially sheltered from the bulk solution by their location within the active site cleft. Probes bound to His 15 have quenching constants about equal to those for the free probes, indicating that this site is highly exposed to the bulk solution.

Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alam, S.Q.; Ren, Y.F.; Alam, B.S.

1987-05-01

The purpose of the present investigation was to determine if dietary lipids can induce changes in the adenylate cyclase system in rat heart. Three groups of male young Sprague-Dawley rats were fed for 6 weeks diets containing 10% corn oil (I), 8% coconut oil + 2% corn oil (II) or 10% menhaden oil (III). Adenylate cyclase activity (basal, fluoride-, isoproterenol-, and forskolin-stimulated) was higher in heart homogenates of rats in group III than in the other two groups. Concentration of the (/sup 3/H)-forskolin binding sites in the cardiac membranes were significantly higher in rats fed menhaden oil. The values (pmol/mgmore » protein) were 4.8 +/- 0.2 (I), 4.5 +/- 0.7 (II) and 8.4 +/- 0.5 (III). There was no significant difference in the affinity of the forskolin binding sites among the 3 dietary groups. When measured at different concentrations of forskolin, the adenylate cyclase activity in cardiac membranes of rats fed menhaden oil was higher than in the other 2 groups. Concentrations of the (/sup 3/H)DHA binding sites were slightly higher but their affinity was lower in cardiac membranes of rats fed menhaden oil. The results suggest that diets containing fish oil increase the concentration of the forskolin binding sites and may also affect the characteristics of the ..beta..-adrenergic receptor in rat heart.« less

Reactivity of a Carbon-Supported Single-Site Molybdenum Dioxo Catalyst for Biodiesel Synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mouat, Aidan R.; Lohr, Tracy L.; Wegener, Evan C.

2016-08-23

A single-site molybdenum dioxo catalyst, (O c) 2Mo(=O) 2@C, was prepared via direct grafting of MoO 2Cl 2(dme) (dme = 1,2-dimethoxyethane) on high-surface- area activated carbon. The physicochemical and chemical properties of this catalyst were fully characterized by N 2 physisorption, ICP-AES/OES, PXRD, STEM, XPS, XAS, temperature-programmed reduction with H 2 (TPR-H 2), and temperature-programmed NH 3 desorption (TPD-NH 3). The single-site nature of the Mo species is corroborated by XPS and TPR-H 2 data, and it exhibits the lowest reported MoO x Tmax of reduction reported to date, suggesting a highly reactive MoVI center. (O c) 2Mo(=O) 2@C catalyzesmore » the transesterification of a variety of esters and triglycerides with ethanol, exhibiting high activity at moderate temperatures (60-90 °C) and with negligible deactivation. (O c) 2Mo(=O) 2@C is resistant to water and can be recycled at least three times with no loss of activity. The transesterification reaction is determined experimentally to be first order in [ethanol] and first order in [Mo] with ΔH = 10.5(8) kcal mol -1 and ΔS = -32(2) eu. The low energy of activation is consistent with the moderate conditions needed to achieve rapid turnover. This highly active carbon-supported single-site molybdenum dioxo species is thus an efficient, robust, and lowcost catalyst with significant potential for transesterification processes.« less

Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.

2012-09-01

The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolutionmore » crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.« less

Evaluation of radioisotope tracer and activation analysis techniques for contamination monitoring in space environment simulation chambers

NASA Technical Reports Server (NTRS)

Smathers, J. B.; Kuykendall, W. E., Jr.; Wright, R. E., Jr.; Marshall, J. R.

1973-01-01

Radioisotope measurement techniques and neutron activation analysis are evaluated for use in identifying and locating contamination sources in space environment simulation chambers. The alpha range method allows the determination of total contaminant concentration in vapor state and condensate state. A Cf-252 neutron activation analysis system for detecting oils and greases tagged with stable elements is described. While neutron activation analysis of tagged contaminants offers specificity, an on-site system is extremely costly to implement and provides only marginal detection sensitivity under even the most favorable conditions.

Impact of Zeolite Aging in Hot Liquid Water on Activity for Acid-Catalyzed Dehydration of Alcohols.

PubMed

Vjunov, Aleksei; Derewinski, Miroslaw A; Fulton, John L; Camaioni, Donald M; Lercher, Johannes A

2015-08-19

The location and stability of Brønsted acid sites catalytically active in zeolites during aqueous phase dehydration of alcohols were studied on the example of cyclohexanol. The catalytically active hydronium ions originate from Brønsted acid sites (BAS) of the zeolite that are formed by framework tetrahedral Si atom substitution by Al. Al K-edge extended X-ray absorption fine structure (EXAFS) and (27)Al magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopies in combination with density functional theory (DFT) calculations are used to determine the distribution of tetrahedral Al sites (Al T-sites) both qualitatively and quantitatively for both parent and HBEA catalysts aged in water prior to catalytic testing. The aging procedure leads to partial degradation of the zeolite framework evidenced from the decrease of material crystallinity (XRD) as well as sorption capacity (BET). With the exception of one commercial zeolite sample, which had the highest concentration of framework silanol-defects, there is no evidence of Al coordination modification after aging in water. The catalyst weight-normalized dehydration rate correlated best with the sum of strong and weak Brønsted acidic protons both able to generate the hydrated hydronium ions. All hydronium ions were equally active for the acid-catalyzed reactions in water. Zeolite aging in hot water prior to catalysis decreased the weight normalized dehydration reaction rate compared to that of the parent HBEA, which is attributed to the reduced concentration of accessible Brønsted acid sites. Sites are hypothesized to be blocked due to reprecipitation of silica dissolved during framework hydrolysis in the aging procedure.

Relative contributions of microbial and infrastructure heat at a crude oil-contaminated site

NASA Astrophysics Data System (ADS)

Warren, Ean; Bekins, Barbara A.

2018-04-01

Biodegradation of contaminants can increase the temperature in the subsurface due to heat generated from exothermic reactions, making temperature observations a potentially low-cost approach for determining microbial activity. For this technique to gain more widespread acceptance, it is necessary to better understand all the factors affecting the measured temperatures. Biodegradation has been occurring at a crude oil-contaminated site near Bemidji, Minnesota for 39 years, creating a quasi-steady-state plume of contaminants and degradation products. A model of subsurface heat generation and transport helps elucidate the contribution of microbial and infrastructure heating to observed temperature increases at this site. We created a steady-state, two-dimensional, heat transport model using previous-published parameter values for physical, chemical and biodegradation properties. Simulated temperature distributions closely match the observed average annual temperatures measured in the contaminated area at the site within less than 0.2 °C in the unsaturated zone and 0.4 °C in the saturated zone. The model results confirm that the observed subsurface heat from microbial activity is due primarily to methane oxidation in the unsaturated zone resulting in a 3.6 °C increase in average annual temperature. Another important source of subsurface heat is from the active, crude-oil pipelines crossing the site. The pipelines impact temperatures for a distance of 200 m and contribute half the heat. Model results show that not accounting for the heat from the pipelines leads to overestimating the degradation rates by a factor of 1.7, demonstrating the importance of identifying and quantifying all heat sources. The model results also highlighted a zone where previously unknown microbial activity is occurring at the site.

Relative contributions of microbial and infrastructure heat at a crude oil-contaminated site.

PubMed

Warren, Ean; Bekins, Barbara A

2018-04-01

Biodegradation of contaminants can increase the temperature in the subsurface due to heat generated from exothermic reactions, making temperature observations a potentially low-cost approach for determining microbial activity. For this technique to gain more widespread acceptance, it is necessary to better understand all the factors affecting the measured temperatures. Biodegradation has been occurring at a crude oil-contaminated site near Bemidji, Minnesota for 39 years, creating a quasi-steady-state plume of contaminants and degradation products. A model of subsurface heat generation and transport helps elucidate the contribution of microbial and infrastructure heating to observed temperature increases at this site. We created a steady-state, two-dimensional, heat transport model using previous-published parameter values for physical, chemical and biodegradation properties. Simulated temperature distributions closely match the observed average annual temperatures measured in the contaminated area at the site within less than 0.2 °C in the unsaturated zone and 0.4 °C in the saturated zone. The model results confirm that the observed subsurface heat from microbial activity is due primarily to methane oxidation in the unsaturated zone resulting in a 3.6 °C increase in average annual temperature. Another important source of subsurface heat is from the active, crude-oil pipelines crossing the site. The pipelines impact temperatures for a distance of 200 m and contribute half the heat. Model results show that not accounting for the heat from the pipelines leads to overestimating the degradation rates by a factor of 1.7, demonstrating the importance of identifying and quantifying all heat sources. The model results also highlighted a zone where previously unknown microbial activity is occurring at the site. Published by Elsevier B.V.

A Great Lakes atmospheric mercury monitoring network: evaluation and design

USGS Publications Warehouse

Risch, Martin R.; Kenski, Donna M.; ,; David, A.

2014-01-01

As many as 51 mercury (Hg) wet-deposition-monitoring sites from 4 networks were operated in 8 USA states and Ontario, Canada in the North American Great Lakes Region from 1996 to 2010. By 2013, 20 of those sites were no longer in operation and approximately half the geographic area of the Region was represented by a single Hg-monitoring site. In response, a Great Lakes Atmospheric Mercury Monitoring (GLAMM) network is needed as a framework for regional collaboration in Hg-deposition monitoring. The purpose of the GLAMM network is to detect changes in regional atmospheric Hg deposition related to changes in Hg emissions. An optimized design for the network was determined to be a minimum of 21 sites in a representative and approximately uniform geographic distribution. A majority of the active and historic Hg-monitoring sites in the Great Lakes Region are part of the National Atmospheric Deposition Program (NADP) Mercury Deposition Network (MDN) in North America and the GLAMM network is planned to be part of the MDN. To determine an optimized network design, active and historic Hg-monitoring sites in the Great Lakes Region were evaluated with a rating system of 21 factors that included characteristics of the monitoring locations and interpretations of Hg data. Monitoring sites were rated according to the number of Hg emissions sources and annual Hg emissions in a geographic polygon centered on each site. Hg-monitoring data from the sites were analyzed for long-term averages in weekly Hg concentrations in precipitation and weekly Hg-wet deposition, and on significant temporal trends in Hg concentrations and Hg deposition. A cluster analysis method was used to group sites with similar variability in their Hg data in order to identify sites that were unique for explaining Hg data variability in the Region. The network design included locations in protected natural areas, urban areas, Great Lakes watersheds, and in proximity to areas with a high density of annual Hg emissions and areas with high average weekly Hg wet deposition. In a statistical analysis, relatively strong, positive correlations in the wet deposition of Hg and sulfate were shown for co-located NADP Hg-monitoring and acid-rain monitoring sites in the Region. This finding indicated that efficiency in regional Hg monitoring can be improved by adding new Hg monitoring to existing NADP acid-rain monitoring sites. Implementation of the GLAMM network design will require Hg-wet-deposition monitoring to be: (a) continued at 12 MDN sites active in 2013 and (b) restarted or added at 9 NADP sites where it is absent in 2013. Ongoing discussions between the states in the Great Lakes Region, the Lake Michigan Air Directors Consortium (a regional planning entity), the NADP, the U.S. Environmental Protection Agency, and the U.S. Geological Survey are needed for coordinating the GLAMM network.

California Dept. of Toxic Substances Control (DTSC) Update of the Schools Naturally Occurring Asbestos Guidance

NASA Astrophysics Data System (ADS)

Malinowski, M.

2012-12-01

Prior to acquisition and/or construction of prospective school sites, the California Education Code mandates that school districts complete environmental assessments and cleanups for prospective new or expanding school sites in order to qualify for state funding. If prospective school sites are determined to have environmental contamination from hazardous materials, including naturally occurring hazardous materials such as naturally occurring asbestos (NOA), where there may be unacceptable potential health risks, the school sites must be properly mitigated prior to occupancy for protection of human health and the environment. NOA is of special concern for schools, because children who are exposed to asbestos may be at increased risk of developing asbestos-related diseases over time. In order to protect human health, the Department of Toxic Substances Control's (DTSC) goals at school sites are to: 1) identify the presence of NOA in school site soils using exposure-reducing soil thresholds; 2) manage potential NOA exposures using mitigation measures to reduce generation of airborne asbestos fibers from soils on school sites; and 3) ensure long-term monitoring and protection of mitigation measures via Operations & Maintenance activities. DTSC is currently in the process of revising its Interim Guidance Naturally Occurring Asbestos (NOA) at School Sites - September 2004. The revisions include: 1) updating the guidance to consider incremental sampling for use at NOA sites in consultation with DTSC's project manager and technical staff, and 2) documenting a tiered approach to addressing high and low activity areas on a school.

Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome.

PubMed

Basu, Debaleena; Kahn, Jennifer N; Li, Xiao-Ping; Tumer, Nilgun E

2016-12-01

The A1 subunits of Shiga toxin 1 (Stx1A1) and Shiga toxin 2 (Stx2A1) interact with the conserved C termini of ribosomal-stalk P-proteins to remove a specific adenine from the sarcin/ricin loop. We previously showed that Stx2A1 has higher affinity for the ribosome and higher catalytic activity than Stx1A1. To determine if conserved arginines at the distal face of the active site contribute to the higher affinity of Stx2A1 for the ribosome, we mutated Arg172, Arg176, and Arg179 in both toxins. We show that Arg172 and Arg176 are more important than Arg179 for the depurination activity and toxicity of Stx1A1 and Stx2A1. Mutation of a single arginine reduced the depurination activity of Stx1A1 more than that of Stx2A1. In contrast, mutation of at least two arginines was necessary to reduce depurination by Stx2A1 to a level similar to that of Stx1A1. R176A and R172A/R176A mutations eliminated interaction of Stx1A1 and Stx2A1 with ribosomes and with the stalk, while mutation of Arg170 at the active site reduced the binding affinity of Stx1A1 and Stx2A1 for the ribosome, but not for the stalk. These results demonstrate that conserved arginines at the distal face of the active site are critical for interactions of Stx1A1 and Stx2A1 with the stalk, while a conserved arginine at the active site is critical for non-stalk-specific interactions with the ribosome. Arginine mutations at either site reduced ribosome interactions of Stx1A1 and Stx2A1 similarly, indicating that conserved arginines are critical for ribosome interactions but do not contribute to the higher affinity of Stx2A1 for the ribosome. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Role of the conserved amino acids of the 'SDN' loop (Ser130, Asp131 and Asn132) in a class A beta-lactamase studied by site-directed mutagenesis.

PubMed

Jacob, F; Joris, B; Lepage, S; Dusart, J; Frère, J M

1990-10-15

Ser130, Asp131 and Asn132 ('SDN') are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.

[Roosting-site characteristics of wintering black-necked cranes (Grus nigricollis) at Napahai, Yunnan].

PubMed

He, Peng; Kong, De-Jun; Liu, Qiang; Yu, Hong-Zhong; Zhao, Jian-Lin; Yang, Xiao-Jun

2011-04-01

From November 2009 to April 2010, roosting-site characteristics of black-necked cranes (Grus nigricollis) were observed at Napahai Provincial Nature Reserve, Shangri-La, Yunnan, China. The positions of roosting-sites were determined by triangulation with markers and field correction. All of the 63 roosting-sites observed were located in patchy marshes with water, which contained some mud on the bottom and 81% of the roosting-sites were covered by plants. They also had a certain distance to areas of human activities and had a certain distance to the shore. A comparison of roosting sites and random sites showed that roosting-sites had thicker mud layers, a higher ratio of open water, longer distance to roads, villages, and farmland, and water depth. Another comparison of before and after usage of roosting-sites found a significant difference in area of marsh patch. Principal component analysis indicated that the usage of roosting-site of black-necked cranes was affected by human disturbance, area of marsh patch, and the condition of the shallow water environment.

Remote Sensing Mars Landing Sites: An Out-of-School Time Planetary Science Education Activity for Middle School Students

NASA Astrophysics Data System (ADS)

Anderson, R. B.; Gaither, T. A.; Edgar, L. A.; Milazzo, M. P.; Vaughan, R. G.; Rubino-Hare, L.; Clark, J.; Ryan, S.

2017-12-01

As part of the Planetary Learning that Advances the Nexus of Engineering, Technology, and Science (PLANETS) project, we have developed an out-of-school time unit for middle school students focused on planetary remote sensing. The activity is divided into two exercises, with the goal of choosing a scientifically interesting and safe landing site for a future Mars mission. Students are introduced to NASA data from several actual and proposed landing sites and must use what they learn about remote sensing to choose a site that satisfies scientific and engineering criteria. The activity also includes background information for educators, including a summary of how landing on Mars helps answer major scientific questions, brief overviews of the data sets that the students will use, summaries of the site geology, and a list of relevant vocabulary. The first exercise introduces students to the concept of reflectance spectroscopy and how it can be used to identify the "fingerprints" of different minerals on the surface of Mars. Students are provided with simplified maps of mineral spectra at the four sites, based on Compact Reconnaissance Imaging Spectrometer (CRISM) observations, as well as a reference sheet with the spectra of common minerals on Mars. They can use this information to determine which sites have hydrated minerals, mafic minerals, or both. The second exercise adds data from the Mars Orbital Laser Altimeter (MOLA), and high resolution visible data from the Context Camera (CTX) on the Mars Reconnaissance Orbiter. Students learn about laser altimetry and how to interpret topographic contours to assess whether a landing site is too rough. The CTX data allow students to study the sites at higher resolution, with annotations that indicate key landforms of interest. These data, along with the spectroscopy data, allow students to rank the sites based on science and engineering criteria. This activity was developed as a collaboration between subject matter experts at the USGS Astrogeology Science Center and education experts from the Northern Arizona University Center for Science Teaching and Learning. It works as either a stand-alone activity or as an extension of the "Worlds Apart" Engineering is Everywhere unit, also developed as part of the PLANETS project in collaboration with the Boston Museum of Science.

Mechanism of activation of methyltransferases involved in translation by the Trm112 'hub' protein.

PubMed

Liger, Dominique; Mora, Liliana; Lazar, Noureddine; Figaro, Sabine; Henri, Julien; Scrima, Nathalie; Buckingham, Richard H; van Tilbeurgh, Herman; Heurgué-Hamard, Valérie; Graille, Marc

2011-08-01

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases.

Mechanism of activation of methyltransferases involved in translation by the Trm112 ‘hub’ protein

PubMed Central

Liger, Dominique; Mora, Liliana; Lazar, Noureddine; Figaro, Sabine; Henri, Julien; Scrima, Nathalie; Buckingham, Richard H.; van Tilbeurgh, Herman; Heurgué-Hamard, Valérie; Graille, Marc

2011-01-01

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases. PMID:21478168

The Remarkable Amphoteric Nature of Defective UiO‐66 in Catalytic Reactions

PubMed Central

Hajek, Julianna; Bueken, Bart; Waroquier, Michel; De Vos, Dirk

2017-01-01

Abstract One of the major requirements in solid acid and base catalyzed reactions is that the reactants, intermediates or activated complexes cooperate with several functions of catalyst support. In this work the remarkable bifunctional behavior of the defective UiO‐66(Zr) metal organic framework is shown for acid‐base pair catalysis. The active site relies on the presence of coordinatively unsaturated zirconium sites, which may be tuned by removing framework linkers and by removal of water from the inorganic bricks using a dehydration treatment. To elucidate the amphoteric nature of defective UiO‐66, the Oppenauer oxidation of primary alcohols has been theoretically investigated using density functional theory (DFT) and the periodic approach. The presence of acid and basic centers within molecular distances is shown to be crucial for determining the catalytic activity of the material. Hydrated and dehydrated bricks have a distinct influence on the acidity and basicity of the active sites. In any case both functions need to cooperate in a concerted way to enable the chemical transformation. Experimental results on UiO‐66 materials of different defectivity support the theoretical observations made in this work. PMID:28736581

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.

Binding sites for interaction of peroxiredoxin 6 with surfactant protein A

PubMed Central

Krishnaiah, Saikumari Y; Dodia, Chandra; Sorokina, Elena M; Li, Haitao; Feinstein, Sheldon I; Fisher, Aron B

2016-01-01

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase and phospholipase A2 (PLA2) activities. This protein participates in the degradation and remodeling of internalized dipalmitoylphosphatidylcholine (DPPC), the major phospholipid component of lung surfactant. We have shown previously that the PLA2 activity of Prdx6 is inhibited by the lung surfactant-associated protein called surfactant protein A (SP-A) through direct protein-protein interaction. Docking of SPA and Prdx6 was modeled using the ZDOCK (zlab.bu.edu) program in order to predict molecular sites for binding of the two proteins. The predicted peptide sequences were evaluated for binding to the opposite protein using isothermal titration calorimetry and circular dichroism measurement followed by determination of the effect of the SP-A peptide on the PLA2 activity of Prdx6. The sequences 195EEEAKKLFPK204.in the Prdx6 helix and 83DEELQTELYEIKHQIL99 in SP-A were identified as the sites for hydrophobic interaction and H+-bonding between the 2 proteins. Treatment of mouse endothelial cells with the SP-A peptide inhibited their recovery from lipid peroxidation associated with oxidative stress indicating inhibition of Prdx6 activity by the peptide in the intact cell. PMID:26723227

The resilience of postglacial hunter-gatherers to abrupt climate change.

PubMed

Blockley, Simon; Candy, Ian; Matthews, Ian; Langdon, Pete; Langdon, Cath; Palmer, Adrian; Lincoln, Paul; Abrook, Ashley; Taylor, Barry; Conneller, Chantal; Bayliss, Alex; MacLeod, Alison; Deeprose, Laura; Darvill, Chris; Kearney, Rebecca; Beavan, Nancy; Staff, Richard; Bamforth, Michael; Taylor, Maisie; Milner, Nicky

2018-05-01

Understanding the resilience of early societies to climate change is an essential part of exploring the environmental sensitivity of human populations. There is significant interest in the role of abrupt climate events as a driver of early Holocene human activity, but there are very few well-dated records directly compared with local climate archives. Here, we present evidence from the internationally important Mesolithic site of Star Carr showing occupation during the early Holocene, which is directly compared with a high-resolution palaeoclimate record from neighbouring lake beds. We show that-once established-there was intensive human activity at the site for several hundred years when the community was subject to multiple, severe, abrupt climate events that impacted air temperatures, the landscape and the ecosystem of the region. However, these results show that occupation and activity at the site persisted regardless of the environmental stresses experienced by this society. The Star Carr population displayed a high level of resilience to climate change, suggesting that postglacial populations were not necessarily held hostage to the flickering switch of climate change. Instead, we show that local, intrinsic changes in the wetland environment were more significant in determining human activity than the large-scale abrupt early Holocene climate events.

A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct.

PubMed

Arjunan, Palaniappa; Sax, Martin; Brunskill, Andrew; Chandrasekhar, Krishnamoorthy; Nemeria, Natalia; Zhang, Sheng; Jordan, Frank; Furey, William

2006-06-02

The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important.

Labeled nucleotide phosphate (NP) probes

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2009-02-03

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Composition for nucleic acid sequencing

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2008-08-26

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Method for sequencing nucleic acid molecules

DOEpatents

Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

2006-06-06

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.