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Sample records for active sites present

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

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-09-18

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

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

    PubMed Central

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

    2012-01-01

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

  3. CHARACTERIZING SITE HYDROLOGY (WORKSHOP MSA PRESENTATION)

    EPA Science Inventory

    Hydrogeology is the foundation of subsurface site characterization for evaluations of monitored natural attenuation (MNA). Three case studies are presented. Examples of the potentially detrimental effects of drilling additives on ground-water samples from monitoring wells are d...

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

    NASA Astrophysics Data System (ADS)

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

    2002-04-01

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

  5. Wind Energy Deployment Process and Siting Tools (Presentation)

    SciTech Connect

    Tegen, S.

    2015-02-01

    Regardless of cost and performance, some wind projects cannot proceed to completion as a result of competing multiple uses or siting considerations. Wind energy siting issues must be better understood and quantified. DOE tasked NREL researchers with depicting the wind energy deployment process and researching development considerations. This presentation provides an overview of these findings and wind siting tools.

  6. Active magnetoplasmonic ruler (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Zubritskaya, Irina

    2016-04-01

    Plasmon rulers are an emerging concept in which the strong near-field coupling of plasmon nanoantenna elements is employed to obtain structural information at the nanoscale. Here, we combine nanoplasmonics and nanomagnetism to conceptualize a magnetoplasmonic dimer nanoantenna that would be able to report nanoscale distances while optimizing its own spatial orientation. The latter constitutes an active operation in which a dynamically optimized optical response per measured unit length allows for the measurement of small and large nanoscale distances with about 2 orders of magnitude higher precision than current state-of-the-art plasmon rulers. We further propose a concept to optically measure the nanoscale response to the controlled application of force with a magnetic field.

  7. Building Toward a Small Wind Turbine Site Assessor Credential (Presentation)

    SciTech Connect

    Sinclair, K.

    2013-09-01

    Proper site assessment is integral to the development of a successful small wind project. Without a small wind site assessor certification program, consumers, including state incentive program managers, lack a benchmark for differentiating between qualified and nonqualified site assessors. A small wind site assessor best practice manual is being developed as a resource for consumers until a credential program becomes available. This presentation describes the purpose, proposed content, and the National Renewable Energy Laboratory's approach to the development of such a manual.

  8. Salt site performance assessment activities

    SciTech Connect

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

    1983-01-01

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

  9. Hanford Site Guidelines for Preparation and Presentation of Geologic Information

    SciTech Connect

    Lanigan, David C.; Last, George V.; Bjornstad, Bruce N.; Thorne, Paul D.; Webber, William D.

    2010-04-30

    A complex geology lies beneath the Hanford Site of southeastern Washington State. Within this geology is a challenging large-scale environmental cleanup project. Geologic and contaminant transport information generated by several U.S. Department of Energy contractors must be documented in geologic graphics clearly, consistently, and accurately. These graphics must then be disseminated in formats readily acceptable by general graphics and document producing software applications. The guidelines presented in this document are intended to facilitate consistent, defensible, geologic graphics and digital data/graphics sharing among the various Hanford Site agencies and contractors.

  10. DNA methylation presents distinct binding sites for human transcription factors.

    PubMed

    Hu, Shaohui; Wan, Jun; Su, Yijing; Song, Qifeng; Zeng, Yaxue; Nguyen, Ha Nam; Shin, Jaehoon; Cox, Eric; Rho, Hee Sool; Woodard, Crystal; Xia, Shuli; Liu, Shuang; Lyu, Huibin; Ming, Guo-Li; Wade, Herschel; Song, Hongjun; Qian, Jiang; Zhu, Heng

    2013-01-01

    DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription. DOI:http://dx.doi.org/10.7554/eLife.00726.001. PMID:24015356

  11. Functional Analyses of Transcription Factor Binding Sites that Differ between Present-Day and Archaic Humans

    PubMed Central

    Weyer, Sven; Pääbo, Svante

    2016-01-01

    We analyze 25 previously identified transcription factor binding sites that carry DNA sequence changes that are present in all or nearly all present-day humans, yet occur in the ancestral state in Neandertals and Denisovans, the closest evolutionary relatives of humans. When the ancestral and derived forms of the transcription factor binding sites are tested using reporter constructs in 3 neuronal cell lines, the activity of 12 of the derived versions of transcription factor binding sites differ from the respective ancestral variants. This suggests that the majority of this class of evolutionary differences between modern humans and Neandertals may affect gene expression in at least some tissue or cell type. PMID:26454764

  12. Site selection: Past and present. [Social planning criteria for Nuclear Power Plant siting

    SciTech Connect

    Tilford, N.R. )

    1994-06-01

    Site selection has been going on since the earliest times. The process has evolved through the Industrial Revolution to the present period of exploding population and environmental awareness. Now the work must be done both with increasing sophistication and greater transparency. Modern techniques for site selection have been developed during the last two decades or so, utilizing a teachable body of knowledge and a growing literature. Many firms and individuals have contributed to this growing field. The driving force has been the need for such a process in siting and licensing of critical facilities such as nuclear power plants. A list of crucial, documented steps for identifying social impacts and acceptability are provided. A recent innovation is the self-selection method developed by government. The Superconducting Supercollider serves as an example of this approach. Geological or geologically dependent factors often dominate the process. The role as engineering and environmental geoscientists is to provide responsible leadership, consultation, and communication to the effort.

  13. Savannah River Site Waste Removal Program - Past, Present and Future

    SciTech Connect

    Saldivar, E.

    2002-02-25

    The Savannah River Site has fifty-one high level waste tanks in various phases of operation and closure. These tanks were originally constructed to receive, store, and treat the high level waste (HLW) created in support of the missions assigned by the Department of Energy (DOE). The Federal Facilities Agreement (FFA) requires the high level waste to be removed from the tanks and stabilized into a final waste form. Additionally, closure of the tanks following waste removal must be completed. The SRS HLW System Plan identifies the interfaces of safe storage, waste removal, and stabilization of the high level waste and the schedule for the closure of each tank. HLW results from the dissolution of irradiated fuel components. Desired nuclear materials are recovered and the byproducts are neutralized with NaOH and sent to the High Level Waste Tank Farms at the SRS. The HLW process waste clarifies in the tanks as the sludge settles, resulting in a layer of dense sludge with salt supernate settling above the sludge. Salt supernate is concentrated via evaporation into saltcake and NaOH liquor. This paper discusses the history of SRS waste removal systems, recent waste removal experiences, and the challenges facing future removal operations to enhance efficiency and cost effectiveness. Specifically, topics will include the evolution and efficiency of systems used in the 1960's which required large volumes of water to current systems of large centrifugal slurry pumps, with significant supporting infrastructure and safety measures. Interactions of this equipment with the waste tank farm operations requirements will also be discussed. The cost and time improvements associated with these present-day systems is a primary focus for the HLW Program.

  14. Hanford Site Beryllium Program: Past, Present, and Future - 12428

    SciTech Connect

    Fisher, Mark; Garcia, Pete; Goeckner, Julie; Millikin, Emily; Stoner, Mike

    2012-07-01

    The U.S. Department of Energy (DOE) has a long history of beryllium use because of the element's broad application to many nuclear operations and processes. At the Hanford Site beryllium alloy was used to fabricate parts for reactors, including fuel rods for the N-Reactor during plutonium production. Because of continued confirmed cases of chronic beryllium disease (CBD), and data suggesting CBD occurs at exposures to low-level concentrations, the DOE decided to issue a rule to further protect federal and contractor workers from hazards associated with exposure to beryllium. When the beryllium rule was issued in 1999, each of the Hanford Site contractors developed a Chronic Beryllium Disease Prevention Program (CBDPP) and initial site wide beryllium inventories. A new site-wide CBDPP, applicable to all Hanford contractors, was issued in May, 2009. In the spring of 2010 the DOE Headquarters Office of Health, Safety, and Security (HSS) conducted an independent inspection to evaluate the status of implementation of the Hanford Site Chronic Beryllium Disease Prevention Program (CBDPP). The report identified four Findings and 12 cross-cutting Opportunities for Improvement (OFIs). A corrective action plan (CAP) was developed to address the Findings and crosscutting OFIs. The DOE directed affected site contractors to identify dedicated resources to participate in development of the CAP, along with involving stakeholders. The CAP included general and contractor-specific recommendations. Following initiation of actions to implement the approved CAP, it became apparent that additional definition of product deliverables was necessary to assure that expectations were adequately addressed and CAP actions could be closed. Consequently, a supplement to the original CAP was prepared and transmitted to DOE-HQ for approval. Development of the supplemental CAP was an eight month effort. From the onset a core group of CAP development members were identified to develop a mechanism for

  15. Auxin Activity: Past, present, and Future1

    PubMed Central

    Enders, Tara A.; Strader, Lucia C.

    2016-01-01

    Long before its chemical identity was known, the phytohormone auxin was postulated to regulate plant growth. In the late 1800s, Sachs hypothesized that plant growth regulators, present in small amounts, move differentially throughout the plant to regulate growth. Concurrently, Charles Darwin and Francis Darwin were discovering that light and gravity were perceived by the tips of shoots and roots and that the stimulus was transmitted to other tissues, which underwent a growth response. These ideas were improved upon by Boysen-Jensen and Paál and were later developed into the Cholodny–Went hypothesis that tropisms were caused by the asymmetric distribution of a growth-promoting substance. These observations led to many efforts to identify this elusive growth-promoting substance, which we now know as auxin. In this review of auxin field advances over the past century, we start with a seminal paper by Kenneth Thimann and Charles Schneider titled “The relative activities of different auxins” from the American Journal of Botany, in which they compare the growth altering properties of several auxinic compounds. From this point, we explore the modern molecular understanding of auxin—including its biosynthesis, transport, and perception. Finally, we end this review with a discussion of outstanding questions and future directions in the auxin field. Over the past 100 yr, much of our progress in understanding auxin biology has relied on the steady and collective advance of the field of auxin researchers; we expect that the next 100 yr of auxin research will likewise make many exciting advances. PMID:25667071

  16. Auxin activity: Past, present, and future.

    PubMed

    Enders, Tara A; Strader, Lucia C

    2015-02-01

    Long before its chemical identity was known, the phytohormone auxin was postulated to regulate plant growth. In the late 1800s, Sachs hypothesized that plant growth regulators, present in small amounts, move differentially throughout the plant to regulate growth. Concurrently, Charles Darwin and Francis Darwin were discovering that light and gravity were perceived by the tips of shoots and roots and that the stimulus was transmitted to other tissues, which underwent a growth response. These ideas were improved upon by Boysen-Jensen and Paál and were later developed into the Cholodny-Went hypothesis that tropisms were caused by the asymmetric distribution of a growth-promoting substance. These observations led to many efforts to identify this elusive growth-promoting substance, which we now know as auxin. In this review of auxin field advances over the past century, we start with a seminal paper by Kenneth Thimann and Charles Schneider titled "The relative activities of different auxins" from the American Journal of Botany, in which they compare the growth altering properties of several auxinic compounds. From this point, we explore the modern molecular understanding of auxin-including its biosynthesis, transport, and perception. Finally, we end this review with a discussion of outstanding questions and future directions in the auxin field. Over the past 100 yr, much of our progress in understanding auxin biology has relied on the steady and collective advance of the field of auxin researchers; we expect that the next 100 yr of auxin research will likewise make many exciting advances.

  17. Catalysis: Elusive active site in focus

    NASA Astrophysics Data System (ADS)

    Labinger, Jay A.

    2016-08-01

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

  18. DESIGN CONSIDERATION INVOLVING ACTIVE SEDIMENT CAPS (PRESENTATION)

    EPA Science Inventory

    When contaminated sediments pose unacceptable risks to human health and the environment, management activities such as removal, treatment, or isolation of contaminated sediments may be required. Various capping designs are being considered for isolating contaminated sediment are...

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

    SciTech Connect

    Not Available

    1990-07-01

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

  20. Active Power Control from Wind Power (Presentation)

    SciTech Connect

    Ela, E.; Brooks, D.

    2011-04-01

    In order to keep the electricity grid stable and the lights on, the power system relies on certain responses from its generating fleet. This presentation evaluates the potential for wind turbines and wind power plants to provide these services and assist the grid during critical times.

  1. 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

  2. Silicosis: a disease with an active present.

    PubMed

    Martínez, Cristina; Prieto, Amador; García, Laura; Quero, Aida; González, Susana; Casan, Pere

    2010-02-01

    Silicosis, an interstitial lung disease caused by the inhalation of crystalline silica powder, despite being one of the oldest occupational diseases, continues being a cause of morbidity and mortality all over the world. The World Health Organisation and the International Labour Organisation (OMS/ILO), aware of the current problem, have designed the World Programme for the Elimination of Silicosis, which includes the identification of occupational groups at risk amongst its actions. We present 3 cases of silicosis in young workers in the construction sector, with exposure to high concentrations of silica due to handling artificial silica conglomerates. The main interest of this observation lies in the identification of new risk sources, in the need to draw attention to the dangers involved in its use without prevention measures, and in the importance of the occupational history to avoid under-diagnosis. PMID:19818543

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

    NASA Astrophysics Data System (ADS)

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

    2006-11-01

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

  4. Active Sites Environmental Monitoring Program: Action levels

    SciTech Connect

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

    1991-10-01

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

  5. Going beyond Lecturing by Using Student Web Site Presentations in a Human Sexuality Course

    ERIC Educational Resources Information Center

    Hughes, Jennifer L.

    2008-01-01

    This paper discusses the use of Web site presentations in human sexuality courses. Web site presentations are a good way to incorporate additional information about human sexuality into a course, and they provide a way to learn to critique human sexuality material on the Internet. In addition, they help students to practice presentation skills. A…

  6. Characterization of active sites in zeolite catalysts

    SciTech Connect

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

    1997-11-01

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

  7. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

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

    1985-01-01

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

  8. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

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

    1992-02-01

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

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

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

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

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

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

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

  11. Using Active Learning Strategies to Present Bloodborne Pathogen Programs

    ERIC Educational Resources Information Center

    Cooper, Leslie; Weaver, Mary G.

    2003-01-01

    Every year, school nurses have the responsibility for developing and presenting a bloodborne pathogen presentation to the education and clerical staff of their buildings. Although the information is similar from year to year, the manner in which the information is presented can be altered. Teachers are using active learning strategies in a variety…

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

    SciTech Connect

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

    2010-08-13

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

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

    SciTech Connect

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

    1993-12-01

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

  14. Sangay volcano, Ecuador: structural development, present activity and petrology

    NASA Astrophysics Data System (ADS)

    Monzier, Michel; Robin, Claude; Samaniego, Pablo; Hall, Minard L.; Cotten, Jo; Mothes, Patricia; Arnaud, Nicolas

    1999-05-01

    Sangay (5230 m), the southernmost active volcano of the Andean Northern Volcanic Zone (NVZ), sits ˜130 km above a >32-Ma-old slab, close to a major tear that separates two distinct subducting oceanic crusts. Southwards, Quaternary volcanism is absent along a 1600-km-long segment of the Andes. Three successive edifices of decreasing volume have formed the Sangay volcanic complex during the last 500 ka. Two former cones (Sangay I and II) have been largely destroyed by sector collapses that resulted in large debris avalanches that flowed out upon the Amazon plain. Sangay III, being constructed within the last avalanche amphitheater, has been active at least since 14 ka BP. Only the largest eruptions with unusually high Plinian columns are likely to represent a major hazard for the inhabited areas located 30 to 100 km west of the volcano. However, given the volcano's relief and unbuttressed eastern side, a future collapse must be considered, that would seriously affect an area of present-day colonization in the Amazon plain, ˜30 km east of the summit. Andesites greatly predominate at Sangay, there being few dacites and basalts. In order to explain the unusual characteristics of the Sangay suite—highest content of incompatible elements (except Y and HREE) of any NVZ suite, low Y and HREE values in the andesites and dacites, and high Nb/La of the only basalt found—a preliminary five-step model is proposed: (1) an enriched mantle (in comparison with an MORB source), or maybe a variably enriched mantle, at the site of the Sangay, prior to Quaternary volcanism; (2) metasomatism of this mantle by important volumes of slab-derived fluids enriched in soluble incompatible elements, due to the subduction of major oceanic fracture zones; (3) partial melting of this metasomatized mantle and generation of primitive basaltic melts with Nb/La values typical of the NVZ, which are parental to the entire Sangay suite but apparently never reach the surface and subordinate

  15. Review of present groundwater monitoring programs at the Nevada Test Site

    SciTech Connect

    Hershey, R.L.; Gillespie, D.

    1993-09-01

    Groundwater monitoring at the Nevada Test Site (NTS) is conducted to detect the presence of radionuclides produced by underground nuclear testing and to verify the quality and safety of groundwater supplies as required by the State of Nevada and federal regulations, and by U.S. Department of Energy (DOE) Orders. Groundwater is monitored at water-supply wells and at other boreholes and wells not specifically designed or located for traditional groundwater monitoring objectives. Different groundwater monitoring programs at the NTS are conducted by several DOE Nevada Operations Office (DOE/NV) contractors. Presently, these individual groundwater monitoring programs have not been assessed or administered under a comprehensive planning approach. Redundancy exists among the programs in both the sampling locations and the constituents analyzed. Also, sampling for certain radionuclides is conducted more frequently than required. The purpose of this report is to review the existing NTS groundwater monitoring programs and make recommendations for modifying the programs so a coordinated, streamlined, and comprehensive monitoring effort may be achieved by DOE/NV. This review will be accomplished in several steps. These include: summarizing the present knowledge of the hydrogeology of the NTS and the potential radionuclide source areas for groundwater contamination; reviewing the existing groundwater monitoring programs at the NTS; examining the rationale for monitoring and the constituents analyzed; reviewing the analytical methods used to quantify tritium activity; discussing monitoring network design criteria; and synthesizing the information presented and making recommendations based on the synthesis. This scope of work was requested by the DOE/NV Hydrologic Resources Management Program (HRMP) and satisfies the 1993 (fiscal year) HRMP Groundwater Monitoring Program Review task.

  16. Active sites in char gasification: Final technical report

    SciTech Connect

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

    1987-09-01

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

  17. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

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

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

  18. Primary Retroperitoneal Melanoma Presented in a Rare Extracutaneous Site for Malignant Melanoma

    PubMed Central

    Alsharedi, Mohamed; Zgheib, Nadim Bou; Khelfa, Yousef; Raufi, Ali; Elmsherghi, Nabiha; Lebowicz, Yehuda

    2016-01-01

    Malignant melanoma, as the name implies, is a malignant tumor of melanocytes, found in the skin, eyes, meningeal lining and the mucosal epithelium of the aero-digestive and genitourinary tracts. Malignant melanoma is typically skin malignancy, which rarely presents at extracutaneous site. Here we present a rare case of primary retroperitoneal melanoma and review the findings in comparison with other cases described in literature. PMID:27746882

  19. Active Sites Environmental Monitoring Program: FY 1991 report

    SciTech Connect

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

    1992-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from April 1991 through September 1991. The ASEMP was established in 1989 by Solid Waste Operations (SWO) and the Environmental Sciences Division, both of Oak Ridge National Laboratory, to provide early detection and performance monitoring at active low-level (radioactive) waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. A new set of action levels was developed on the basis of a statistical analysis of background contamination. These new action levels have been used to evaluate results in this report. Results of ASEMP monitoring continue to demonstrate that no LLW (except [sup 3]H) is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II, which began in early FY 1991, was >90% complete at the end of September 1991. Results of sampling of groundwater and surface waters is presented.

  20. Inhibition and active-site modelling of prolidase.

    PubMed

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

    1989-03-15

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

  1. OceanSITES format and Ocean Observatory Output harmonisation: past, present and future

    NASA Astrophysics Data System (ADS)

    Pagnani, Maureen; Galbraith, Nan; Diggs, Stephen; Lankhorst, Matthias; Hidas, Marton; Lampitt, Richard

    2015-04-01

    initiative has always been truly international, and in Europe the first project to include OceanSITES as part of its outputs was ANIMATE(2002-2005), where 3 moorings and 5 partners shared equipment, methods and analysis effort and produced their final outputs in OceanSITES format. Subsequent European projects, MERSEA(2004-2008) and EuroSITES (2008-2011) built on that early success and the current European project FixO3 encompasses 23 moorings and 29 partners, all of whom are committed to producing data in OceanSITES format. The global OceanSITES partnership continues to grow; in 2014 the Australian Integrated Marine Observing System ( IMOS) started delivering data to the OceanSITES FTP, and files and India, South Korea and Japan are also active members of the OceanSITES community. As illustrated in figure 1 the OceanSITES sites cover the entire globe, and the format has now matured enough to be taken up by other user groups. GO-SHIP, a global, ship-based hydrographic program, shares technical management with OceanSITES through JCOMMOPS, and has its roots in WOCE Hydrography. This program complements OceanSITES and directly contributes to the mooring data holdings by providing repeated CTD and bottle profiles at specific locations. GO-SHIP hydrographic data adds a source of timeseries profiles and are provided in the OceanSITES file structure to facilitate full data interoperability. GO-SHIP has worked closely with the OceanSITES program, and this interaction has produced an unexpected side benefit - all data in the GO-SHIP database will be offered the robust and CF-compliant OceanSITES format beginning in 2015. The MyOcean European ocean monitoring and forecasting project has been in existence since 2009, and has successfully used the OceanSITES format as a unifying paradigm. MyOcean daily receives hundreds of data files from across Europe, and distributes the data from drifter buoys, moorings and tide gauges in OceanSITES format. These in-situ data are essential for both

  2. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  3. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  4. OceanSITES format and Ocean Observatory Output harmonisation: past, present and future

    NASA Astrophysics Data System (ADS)

    Pagnani, Maureen; Galbraith, Nan; Diggs, Stephen; Lankhorst, Matthias; Hidas, Marton; Lampitt, Richard

    2015-04-01

    initiative has always been truly international, and in Europe the first project to include OceanSITES as part of its outputs was ANIMATE(2002-2005), where 3 moorings and 5 partners shared equipment, methods and analysis effort and produced their final outputs in OceanSITES format. Subsequent European projects, MERSEA(2004-2008) and EuroSITES (2008-2011) built on that early success and the current European project FixO3 encompasses 23 moorings and 29 partners, all of whom are committed to producing data in OceanSITES format. The global OceanSITES partnership continues to grow; in 2014 the Australian Integrated Marine Observing System ( IMOS) started delivering data to the OceanSITES FTP, and files and India, South Korea and Japan are also active members of the OceanSITES community. As illustrated in figure 1 the OceanSITES sites cover the entire globe, and the format has now matured enough to be taken up by other user groups. GO-SHIP, a global, ship-based hydrographic program, shares technical management with OceanSITES through JCOMMOPS, and has its roots in WOCE Hydrography. This program complements OceanSITES and directly contributes to the mooring data holdings by providing repeated CTD and bottle profiles at specific locations. GO-SHIP hydrographic data adds a source of timeseries profiles and are provided in the OceanSITES file structure to facilitate full data interoperability. GO-SHIP has worked closely with the OceanSITES program, and this interaction has produced an unexpected side benefit - all data in the GO-SHIP database will be offered the robust and CF-compliant OceanSITES format beginning in 2015. The MyOcean European ocean monitoring and forecasting project has been in existence since 2009, and has successfully used the OceanSITES format as a unifying paradigm. MyOcean daily receives hundreds of data files from across Europe, and distributes the data from drifter buoys, moorings and tide gauges in OceanSITES format. These in-situ data are essential for both

  5. 17 CFR 229.1206 - (Item 1206) Present activities.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ..., the registrant's present activities, such as the number of wells in the process of being drilled... files the document as reasonably possible. (c) Include only those wells in the process of being drilled... ENERGY POLICY AND CONSERVATION ACT OF 1975-REGULATION S-K Disclosure by Registrants Engaged in Oil...

  6. Dynamical downscaling of present climate extremal episodes for the BINGO research site of Cyprus

    NASA Astrophysics Data System (ADS)

    Zittis, George; Hadjinicolaou, Panos; Bruggeman, Adriana; Camera, Corrado; Lelieveld, Jos

    2016-04-01

    Besides global warming, climate change is expected to cause alterations in precipitation amounts and distribution than can be linked to extreme events such as floods or prolonged droughts. This will have a significant impact in strategic societal sectors that base their activities on water resources. While the global climate projections inform us about the long-term and weather forecasts can give useful information only for a few days or weeks, decision-makers and end-users also need guidance on inter-annual to decadal time scales. In this context, the BINGO (Bringing INnovation to onGOing water management - a better future under climate change) H2020 project aims both at reducing the uncertainty of near-term climate predictions and developing response strategies in order to better manage the remaining uncertainty. One of the project's main objectives is to develop improved decadal predictions, in adequate spatiotemporal scales, with a specific focus on extreme precipitation events. The projected rainfall will be eventually used to drive hydrological impact models. BINGO focuses on research sites that encompass river basins, watersheds and urban areas of six European countries including Norway, Cyprus, Germany, Portugal, The Netherlands and Spain. In this study we present the dynamical downscaling of the ERA-Interim dataset for validation purposes and for the research site of Cyprus. Five extreme rainfall periods were identified from the observed precipitation archives and were simulated in very high horizontal resolutions (4~1 km) using the WRF limited area atmospheric model. To optimize the performance of the model we have tested a combination of three cumulus and five microphysics parameterization schemes that resulted in 15 simulations for each extreme precipitation event. The model output was compared with daily or hourly (where available) representative rain gauge data. A set of statistical metrics was applied in order to objectively select the best

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

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

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

    DOE Data Explorer

    John Peterson

    2015-04-17

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

  10. The Earliest Presenting Umbilical Port Site Hernia Following Laparoscopic Cholecystectomy: A Case Report

    PubMed Central

    Sharma, Rajeev; Goyal, Manav; Gupta, Sanjay

    2016-01-01

    Port site hernia after laparoscopic surgery is a rare complication. Here we present a case of a 55-year-old female, diagnosed with an anterior abdominal wall hernia through the 10mm umbilical port, just two days after her laparoscopic cholecystectomy. The uniqueness of this case is its extremely early presentation. Patient presented with features of acute intestinal obstruction and due to prompt diagnosis and timely intervention, she underwent a successful reduction of hernia and an anatomical repair of the fascial and peritoneal defect through the midline laparotomy incision.

  11. The Earliest Presenting Umbilical Port Site Hernia Following Laparoscopic Cholecystectomy: A Case Report

    PubMed Central

    Sharma, Rajeev; Goyal, Manav; Gupta, Sanjay

    2016-01-01

    Port site hernia after laparoscopic surgery is a rare complication. Here we present a case of a 55-year-old female, diagnosed with an anterior abdominal wall hernia through the 10mm umbilical port, just two days after her laparoscopic cholecystectomy. The uniqueness of this case is its extremely early presentation. Patient presented with features of acute intestinal obstruction and due to prompt diagnosis and timely intervention, she underwent a successful reduction of hernia and an anatomical repair of the fascial and peritoneal defect through the midline laparotomy incision. PMID:27630904

  12. The Earliest Presenting Umbilical Port Site Hernia Following Laparoscopic Cholecystectomy: A Case Report.

    PubMed

    Sharma, Rajeev; Mehta, Deeksha; Goyal, Manav; Gupta, Sanjay

    2016-07-01

    Port site hernia after laparoscopic surgery is a rare complication. Here we present a case of a 55-year-old female, diagnosed with an anterior abdominal wall hernia through the 10mm umbilical port, just two days after her laparoscopic cholecystectomy. The uniqueness of this case is its extremely early presentation. Patient presented with features of acute intestinal obstruction and due to prompt diagnosis and timely intervention, she underwent a successful reduction of hernia and an anatomical repair of the fascial and peritoneal defect through the midline laparotomy incision. PMID:27630904

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

    SciTech Connect

    Hartman, F.C.

    1991-01-01

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

  14. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

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

  15. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

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

  16. Biological Activities of Phenolic Compounds Present in Virgin Olive Oil

    PubMed Central

    Cicerale, Sara; Lucas, Lisa; Keast, Russell

    2010-01-01

    The Mediterranean diet is associated with a lower incidence of atherosclerosis, cardiovascular disease, neurodegenerative diseases and certain types of cancer. The apparent health benefits have been partially ascribed to the dietary consumption of virgin olive oil by Mediterranean populations. Much research has focused on the biologically active phenolic compounds naturally present in virgin olive oils to aid in explaining reduced mortality and morbidity experienced by people consuming a traditional Mediterranean diet. Studies (human, animal, in vivo and in vitro) have demonstrated that olive oil phenolic compounds have positive effects on certain physiological parameters, such as plasma lipoproteins, oxidative damage, inflammatory markers, platelet and cellular function, antimicrobial activity and bone health. This paper summarizes current knowledge on the bioavailability and biological activities of olive oil phenolic compounds. PMID:20386648

  17. Initial Presentation Sites as Predictors of Herpes Zoster Complications: A Nationwide Cohort Study

    PubMed Central

    Wang, Wen-Yi; Liu, Sing-Huh; Lin, Meng-Yin; Lin, Che-Chen

    2016-01-01

    Herpes zoster (HZ) is associated with complications such as postherpetic neuralgia (PHN) and HZ ophthalmicus (HZO). However, few studies have focused on identifying patients having a high risk of PHN and HZO according to the initial presentation sites. The current study investigated these factors in a nationwide population-based cohort derived from Taiwan’s Longitudinal Health Insurance Database. The results indicate that the initial presentation sites can predict the complication site of HZ. In this study, elderly patients were found to be more susceptible to HZ and were the first to present with neurological signs (HZN). Furthermore, compared with patients with HZO and other signs (HZT), those with HZN had a higher comorbidity risk. Patients with HZN showed a significantly higher visceral complication risk than did those with HZO (adjusted hazard ratio [HR] = 1.47, 95% confidence interval [CI] = 1.27–1.71). In addition, patients with HZT showed lower risks of ocular and neurological complications than did those with HZN after stratification by age and sex (adjusted HR = 0.46, 95% CI = 0.31–0.68 and HR = 0.73, 95% CI = 0.59–0.91, respectively). PMID:27711168

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

    PubMed Central

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

    1997-01-01

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

  19. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

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

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

    SciTech Connect

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

    2010-09-07

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

  1. Drug eruptions presenting at sites of prior radiation damage (sunlight and electron beam)

    SciTech Connect

    Shelley, W.B.; Shelley, E.D.; Campbell, A.C.; Weigensberg, I.J.

    1984-07-01

    Two patients are described in whom sunburn and electron beam radiodermatitis, respectively, were critical determinants in localizing the initial presentation of drug eruptions. In the first instance, a severe sunburn of the back and thighs was followed 7 months later by the appearance of a toxic epidermal necrolysis drug reaction to trimethoprim-sulfamethoxazole in the exact sites of the previous bullous sunburn reaction. In the second patient, a radiodermatitis of the left upper arm due to electron beam therapy for metastatic breast cancer was followed 7 weeks later by a codeine drug reaction confined to the area of the radiodermatitis. In both instances, oral rechallenge with the offending drug reproduced the eruption.

  2. Characterization of the methanotrophic bacterial community present in a trichloroethylene-contaminated subsurface groundwater site.

    PubMed Central

    Bowman, J P; Jiménez, L; Rosario, I; Hazen, T C; Sayler, G S

    1993-01-01

    Groundwater, contaminated with trichloroethylene (TCE) and tetrachloroethylene (PCE), was collected from 13 monitoring wells at Area M on the U.S. Department of Energy Savannah River Site near Aiken, S.C. Filtered groundwater samples were enriched with methane, leading to the isolation of 25 methanotrophic isolates. The phospholipid fatty acid profiles of all the isolates were dominated by 18:1 omega 8c (60 to 80%), a signature lipid for group II methanotrophs. Subsequent phenotypic testing showed that most of the strains were members of the genus Methylosinus and one isolate was a member of the genus Methylocystis. Most of the methanotroph isolates exhibited soluble methane monooxygenase (sMMO) activity. This was presumptively indicated by the naphthalene oxidation assay and confirmed by hybridization with a gene probe encoding the mmoB gene and by cell extract assays. TCE was degraded at various rates by most of the sMMO-producing isolates, whereas PCE was not degraded. Savannah River Area M and other groundwaters, pristine and polluted, were found to support sMMO activity when supplemented with nutrients and then inoculated with Methylosinus trichosporium OB3b. The maximal sMMO-specific activity obtained in the various groundwaters ranged from 41 to 67% compared with maximal rates obtained in copper-free nitrate mineral salts media. This study partially supports the hypothesis that stimulation of indigenous methanotrophic communities can be efficacious for removal of chlorinated aliphatic hydrocarbons from subsurface sites and that the removal can be mediated by sMMO. PMID:8368829

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

    PubMed

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

    2015-07-21

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

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

    SciTech Connect

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

    1986-03-20

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

  5. Taiwan's cooperative space activities at present and in future

    NASA Astrophysics Data System (ADS)

    Ip, Wing-Huen

    2004-01-01

    Taiwan is developing a long-term space program which is entering its next 15 years of planning cycle. Since its establishment in 1992, the National Space Program Office has played a key role in introducing satellite technology and space experiments into Taiwan. In parallel, basic research in space science and remote-sensing observations are being promoted in different institutions. A combination of these efforts has earned Taiwan a compact but effective space program capable of mounting satellite missions and advanced study in various disciplines of space science. The satellite data receiving and data processing facilities are particularly valuable in addressing issues related to environmental protection, natural hazards and economic planning. At the present time, Taiwan's international cooperative space activities are still very limited in scope but there is a wide ranging of possibilities which could be pursued together with other developing nations in space research under the auspice of COSPAR.

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

    PubMed

    Chou, Kuo-Chen; Cai, Yu-dong

    2004-04-01

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

  7. Activation of cells using femtosecond laser beam (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Batabyal, Subrata; Satpathy, Sarmishtha; Kim, Young-tae; Mohanty, Samarendra K.

    2016-03-01

    Study of communication in cellular systems requires precise activation of targeted cell(s) in the network. In contrast to chemical, electrical, thermal, mechanical stimulation, optical stimulation is non-invasive and is better suited for stimulation of targeted cells. As compared to visible lasers, the near infrared (NIR) microsecond/nanosecond pulsed laser beams are being used as preferred stimulation tool as they provide higher penetration depth in tissues. Femotosecond (FS) laser beams in NIR are also being used for direct and indirect (i.e. via two-photon optogenetics) stimulation of cells. Here, we present a comparative evaluation of efficacy of NIR FS laser beam for direct (no optogenetic sensitization) and 2ph optogenetic stimulation of cells. Further, for the first time, we demonstrate the use of blue (~450 nm, obtained by second harmonic generation) FS laser beam for stimulation of cells with and without Channelrhodopisn-2 (ChR2) expression. Comparative analysis of photocurrent generated by blue FS laser beam and continuous wave blue light for optogenetics stimulation of ChR2 transfected HEK cells will be presented. The use of ultrafast laser micro-beam for focal, non-contact, and repeated stimulation of single cells in a cellular circuitry allowed us to study the communication between different cell types.

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

    SciTech Connect

    Miao, Yinglong; Baudry, Jerome Y

    2011-01-01

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

  9. A small ribozyme with dual-site kinase activity

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  11. Present Day Activity of South Polar Gullies on Mars

    NASA Astrophysics Data System (ADS)

    Raack, J.; Reiss, D.; Ruesch, O.; Hiesinger, H.

    2012-04-01

    Here we report on clearly identified seasonal changes of gullies observed within the last two martian years (MY) on slopes of a south polar pit, which is located in a filled crater (diameter ~54 km) north of Sisyphi Cavi at ~68.5°S and ~1.5°E. Using new high-resolution imaging (High Resolution Imaging Science Experiment, HiRISE), temperature (Thermal Emission Spectrometer, TES) and spectral data (Compact Reconnaissance Imaging Spectrometer for Mars, CRISM; Observatoire pour la Minéralogie, l'Eau, les Glaces et l'Activité, OMEGA), we analyzed the exact timing of changes of gullies and detect the possible medium (CO2, H2O or dry) and mechanism which initiate present day gully activity. Two locations in the study region with clear modifications of gullies were identified in MY 29 between LS 226° and LS 247° and between LS 209° and LS 247°. In MY 30 changes occur in both locations between LS 218° and LS 249°. Modifications are the formation of a new small apron and new deposits within the channel, both associated with the deposition of dark material. Erosion in gully alcoves or channels was not observed. TES data show temperatures between ~180 and ~240 K within the period of gully modifications. Maximum temperatures in the region rise up to ~285 K between LS ~270° and ~310°. Spectral data show a CO2-cover of the study region until LS 227°. CO2-ice free surface are spectrally observed for the first time at LS 249°. H2O was not spectrally detected in the study region and a mixture of CO2 and H2O as presented in [1] cannot be clearly detected. Unfortunately, there are no spectral data available between LS 227° and 249°. Modifications of gullies imply seasonal volatile activity. The activity can be narrowed down to occur between LS 226° and 247° at mean temperatures between ~180 and ~240 K. This is in the range of temperatures where CO2 sublimates back into the atmosphere. Based on the temperature range, the most likely candidate for the observed new

  12. Merkel cell carcinoma of unknown primary site; case presentation and review of the literature.

    PubMed

    Kontis, Elissaios; Vezakis, Antonios; Pantiora, Eirini; Stasinopoulou, Sotiria; Polydorou, Andreas; Voros, Dionysios; Fragulidis, Georgios P

    2015-12-01

    Merkel cell carcinoma (MCC) is a rare skin malignancy associated with sun exposure and considered as a Neuroendocrine Tumor due to its characteristic histologic features. However there is increasing number of reports of Unknown Primary MCC's (UPMCC). Although initially UPMCC was considered a variant of known primary MCC, there is growing evidence that it could represent a different clinical entity. We present the case of a 60 year-old male patient who was referred to our department for surgical management of lymph node disease for UPMCC. The patient had undergone excisional biopsy of an inguinal lump, which was found to be an infiltrated lymph node by MCC. The patient underwent full imaging staging including a PET/CT, which failed to identify a primary site, and revealed only intra-abdominal lymph node disease. The patient underwent extended retroperitoneal and inguinal lymph node dissection and remains free of recurrence 16 months postoperatively. PMID:26904196

  13. Photovoltaic Reliability Group activities in USA and Brazil (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    Dhere, Neelkanth G.; Cruz, Leila R. O.

    2015-09-01

    Recently prices of photovoltaic (PV) systems have been reduced considerably and may continue to be reduced making them attractive. If these systems provide electricity over the stipulated warranty period, it would be possible attain socket parity within the next few years. Current photovoltaic module qualifications tests help in minimizing infant mortality but do not guarantee useful lifetime over the warranty period. The PV Module Quality Assurance Task Force (PVQAT) is trying to formulate accelerated tests that will be useful towards achieving the ultimate goal of assuring useful lifetime over the warranty period as well as to assure manufacturing quality. Unfortunately, assuring the manufacturing quality may require 24/7 presence. Alternatively, collecting data on the performance of fielded systems would assist in assuring manufacturing quality. Here PV systems installed by home-owners and small businesses can constitute as an important untapped source of data. The volunteer group, PV - Reliable, Safe and Sustainable Quality! (PVRessQ!) is providing valuable service to small PV system owners. Photovoltaic Reliability Group (PVRG) is initiating activities in USA and Brazil to assist home owners and small businesses in monitoring photovoltaic (PV) module performance and enforcing warranty. It will work in collaboration with small PV system owners, consumer protection agencies. Brazil is endowed with excellent solar irradiance making it attractive for installation of PV systems. Participating owners of small PV systems would instruct inverter manufacturers to copy the daily e-mails to PVRG and as necessary, will authorize the PVRG to carry out review of PV systems. The presentation will consist of overall activities of PVRG in USA and Brazil.

  14. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  15. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  16. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

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

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

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

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

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

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

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

  19. Life detection at a Mars analogue site of present-day serpentinization in the Tablelands Ophiolite of Newfoundland (Invited)

    NASA Astrophysics Data System (ADS)

    Morrill, P. L.; Szponar, N.; Brazelton, W. J.; Woodruff, Q.; Schrenk, M. O.; Bower, D. M.; Steele, A.

    2010-12-01

    The Tableland Ophiolite was created during the collision of Laurentia and Gondwana continents ca. 470 million years ago. Ultramafic mantle rocks, from the ancient sea bed that once separated these continents, were thrusted westward onto the old continental margin, which is now Western Newfoundland. Weathering due to recent glaciations has left large areas of ultramafic rock at the surface and created fissures for fluid flow. As a result serpentinization is occurring as fresh water penetrates the unaltered ultramafic rock. Serpentinization is of particular interest because, through hydration of ultramafic rock, this reaction produces H2 and the reducing conditions necessary for abiogenic hydrocarbon synthesis, while also producing conditions amenable for chemolithotrophic life. Therefore sites of active serpentinization can be the source of either abiogenic or biogenic organics, or both. Serpentinization is a suspected (past or present) source of (detected or putative) hydrocarbons on Mars, Titan and Europa, hence these astrobodies may be potentially habitable or once habitable environments. The Tablelands Ophiolite is an analogue site that is ideal for testing methods of life detection in an extreme environment of high pH and low microbial biomass characteristic of sites of serpentinization. Multiple ultrabasic reducing springs characteristic of present-day serpentinization have been identified and characterized based on their geochemistry and microbiology. Field-based instruments were deployed for the detection of microbial activity (ATP), microbial cell wall material, and mineralogy, in yet untested high pH and low biomass environment. In this talk I will give an overview of the in situ measurements of life detection and put these measurements in context of geochemistry, microbiology, carbon source and reaction pathways, and I will discuss what we have learned that will help us plan for future mission measurements.

  20. O-glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1.

    PubMed

    Rana, Nadia A; Nita-Lazar, Aleksandra; Takeuchi, Hideyuki; Kakuda, Shinako; Luther, Kelvin B; Haltiwanger, Robert S

    2011-09-01

    Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both. Here we examine the occupancy of the predicted O-glucose sites on mouse Notch1 (mN1) using the consensus sequence C(1)XSXPC(2). We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site- and cell type-dependent. For instance, although most sites are modified at high stoichiometries, the site at EGF 27 is only partially glucosylated, and the occupancy of the site at EGF 4 varies with cell type. O-Glucose is also found at a novel, non-traditional consensus site at EGF 9. Based on this finding, we propose a revision of the consensus sequence for O-glucosylation to allow alanine N-terminal to cysteine 2: C(1)XSX(A/P)C(2). We also show through biochemical and mass spectral analyses that serine is the only hydroxyamino acid that is modified with O-glucose on EGF repeats. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc. To establish the functional importance of individual O-glucose sites in mN1, we used a cell-based signaling assay. Elimination of most individual sites shows little or no effect on mN1 activation, suggesting that the major effects of O-glucose are mediated by modification of multiple sites. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch, does significantly reduce activity. These results demonstrate that, like O-fucose, the O-glucose modifications of EGF repeats occur extensively on mN1, and they play important roles in Notch function.

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

    PubMed

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

    2012-09-01

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

  2. Solitary fibrous tumor: histological and immunohistochemical spectrum of benign and malignant variants presenting at different sites.

    PubMed

    Hanau, C A; Miettinen, M

    1995-04-01

    Twenty-nine tumors (from 26 patients, including two with recurrent disease) diagnosed as solitary fibrous tumor (SFT) of the pleura (n = 23), mediastinum (n = 4), abdominal cavity (n = 1), and parotid gland (n = 1) were studied immunohistochemically. Three histologically malignant tumors showed areas of high cellularity and mitotic activity (more than 4 mitoses/10 high-power fields) with features resembling malignant fibrous histiocytoma, malignant hemangiopericytoma, or fibrosarcoma, together with areas typical of benign solitary fibrous tumor. Formaldehyde-fixed, paraffin-embedded tissues and avidin-biotin-complex immunostaining were used. All of the tumors showed vimentin positivity and did not stain for cytokeratin, glial fibrillary acidic protein, or muscle cell markers, except for focal desmin reactivity in seven tumors, mostly seen in frozen sections, and focal keratin reactivity in one histologically malignant tumor. The neoplastic cells were positive for CD34 and negative for CD31; these patterns also were seen in the three histologically malignant cases. In nine of the cases acetone-fixed frozen sections showed variable focal positivity for neurofilament proteins of 68 kd. We conclude that SFT is a neoplasm of fibroblasts/primitive mesenchymal cells with features of multidirectional differentiation. We also report the finding of a novel site for SFT, the parotid gland.

  3. Scale-free brain activity: past, present and future

    PubMed Central

    He, Biyu J.

    2014-01-01

    Brain activity observed at many spatiotemporal scales exhibits a 1/f-like power spectrum, including neuronal membrane potentials, neural field potentials, noninvasive electroencephalography, magnetoencephalography and functional magnetic resonance imaging signals. A 1/f-like power spectrum is indicative of arrhythmic brain activity that does not contain a predominant temporal scale (hence, “scale-free”). This characteristic of scale-free brain activity distinguishes it from brain oscillations. While scale-free brain activity and brain oscillations coexist, our understanding of the former remains very limited. Recent research has shed light on the spatiotemporal organization, functional significance and potential generative mechanisms of scale-free brain activity, as well as its developmental and clinical relevance. A deeper understanding of this prevalent brain signal should provide new insights and analytical tools for cognitive neuroscience. PMID:24788139

  4. Scale-free brain activity: past, present, and future.

    PubMed

    He, Biyu J

    2014-09-01

    Brain activity observed at many spatiotemporal scales exhibits a 1/f-like power spectrum, including neuronal membrane potentials, neural field potentials, noninvasive electroencephalography (EEG), magnetoencephalography (MEG), and functional magnetic resonance imaging (fMRI) signals. A 1/f-like power spectrum is indicative of arrhythmic brain activity that does not contain a predominant temporal scale (hence, 'scale-free'). This characteristic of scale-free brain activity distinguishes it from brain oscillations. Although scale-free brain activity and brain oscillations coexist, our understanding of the former remains limited. Recent research has shed light on the spatiotemporal organization, functional significance, and potential generative mechanisms of scale-free brain activity, as well as its developmental and clinical relevance. A deeper understanding of this prevalent brain signal should provide new insights into, and analytical tools for, cognitive neuroscience.

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

    SciTech Connect

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

    1991-10-01

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

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

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    1989-01-01

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

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

    SciTech Connect

    Kefang Jiao; Song Li; Zhengzheng Lu

    1996-12-31

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  10. The Wahluke (North) Slope of the Hanford Site: History and present challenges

    SciTech Connect

    Gerber, M.S.

    1996-04-16

    The Hanford Site was founded in early 1943 for the top secret government mission of producing plutonium for the world`s first atomic weapons. A great deal of land was needed, both to separate various Site facilities from each other, and to provide buffer zones for safety and security purposes. In total, 640 square miles were occupied by the original Hanford Site and its buffer zones. Much of this land had been earmarked for inclusion in the Columbia Basin Irrigation Project (CBP). After World War II ended, a series of national decisions led to a long-term mission for the Hanford Site, and area residents learned that the Site lands they had hoped to farm would be withheld from agricultural production for the foreseeable future. A long set of negotiations commenced between the federal management agency responsible for Hanford (the Atomic Energy Commission -- AEC), and the Bureau of Reclamation (BOR), Department of the Interior that managed the CBP. Some lands were turned back to agriculture, and other compromises made, in the Site`s far northern buffer lands known as the Wahluke Slope, during the 1950s. In the mid-1960s, further negotiations were about to allow farming on lands just north of the Columbia River, opposite Hanford`s reactors, when studies conducted by the BOR found drainage barriers to irrigation. As a result of these findings, two wildlife refuges were created on that land in 1971. Today, after the Hanford Site plutonium production mission has ended and as Site cleanup goes forward, the possibility of total release of Wahluke Slope lands from the control of the Department of Energy (DOE -- a successor agency to the AEC) is under discussion. Such discussion encompasses not just objective and clearly visible criteria, but it resurrects historical debates about the roles of farming and government presence in the Columbia Basin.

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

    PubMed Central

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

    2008-01-01

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

  12. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

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

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

    PubMed

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

    2014-12-16

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

  14. Activated Brain Endothelial Cells Cross-Present Malaria Antigen

    PubMed Central

    Howland, Shanshan W.; Poh, Chek Meng; Rénia, Laurent

    2015-01-01

    In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention. PMID:26046849

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

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

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

  16. Energy transfer at the active sites of heme proteins

    SciTech Connect

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

    1995-12-31

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

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

    PubMed

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

    2016-09-01

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

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

    PubMed Central

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

    2010-01-01

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

  19. Activities at Fermilab related to collider present and future

    NASA Astrophysics Data System (ADS)

    Goderre, G. P.; Holt, J.

    1992-11-01

    The long-range Fermilab program requires fully capitalizing on the world's highest energy accelerator, the Tevatron, throughout the decade of the 90's. The program calls for increasing the collider luminosity with each successive run until peak luminosities of ≳5×1031 cm-2 s-1 and integrated luminosities of ≳100 pb-1 per run are achieved, effectively doubling the mass range accessible for discovery. If the quark lies at the upper range of the mass of the Tevatron, then increasing the energy of the collider operation could prove to be a crucial factor in the future program as well. In order to achieve these goals, we present a highly challenging upgrade of the present accelerator complex, called Fermilab III. In order to increase this performance level by a factor of 50, many changes are needed. Such a plan, of necessity, has modifications in almost all areas of the accelerator as the present system is reasonably optimized. (AIP)

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

    PubMed

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

    1990-03-01

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

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

    PubMed Central

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

    2015-01-01

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

  2. Present and Future Activities on Neutron Imaging in Argentina

    NASA Astrophysics Data System (ADS)

    Tartaglione, Aureliano; Blostein, Jerónimo; Cantargi, Florencia; Marín, Julio; Baruj, Alberto; Meyer, Gabriel; Santisteban, Javier; Sánchez, Fernando

    We present here a short review of the main work which has been done in the latest years in neutron imaging in Argentina, and the future plans for the development of this technique in the country, mainly focused in the design of a new neutron imaging instrument to be installed in the future research reactor RA10. We present here the results of the implementation of the technique in samples belonging to the Argentinean cultural heritage and experiments related with hydrogen storage. At the same time, the Argentinean RA10 project for the design and construction of a 30 MW multipurpose research reactor is rapidly progressing. It started to be designed by the National Atomic Energy Commission (CNEA) and the technology company INVAP SE, both from Argentina, in June 2010. The construction will start in the beginning of 2015 in the Ezeiza Atomic Center, at 36 km from Buenos Aires City, and is expected to be finished by 2020. One of the main aims of the project is to offer to the Argentinean scientific and technology system new capabilities based on neutron techniques. We present here the conceptual design of a neutron imaging facility which will use one of the cold neutron beams, and will be installed in the reactor hall. Preliminary simulation results show that at the farthest detection position, at about 17 m from the cold source, a uniform neutron beam on a detection screen with an intensity of about 108 n/cm2/s is expected.

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

    SciTech Connect

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

    1993-12-01

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

  4. Interfering with mineralocorticoid receptor activation: the past, present, and future

    PubMed Central

    2014-01-01

    Aldosterone is a potent mineralocorticoid produced by the adrenal gland. Aldosterone binds to and activates the mineralocorticoid receptor (MR) in a plethora of tissues, but the cardiovascular actions of aldosterone are of primary interest clinically. Although MR antagonists were developed as antihypertensive agents, they are now considered to be important therapeutic options for patients with heart failure. Specifically, blocking only the MR has proven to be a difficult task because of its similarity to other steroid receptors, including the androgen and progesterone receptors. This lack of specificity caused the use of the first-generation mineralocorticoid receptor antagonists to be fraught with difficulty because of the side effects produced by drug administration. However, in recent years, several advances have been made that could potentially increase the clinical use of agents that inhibit the actions of aldosterone. These will be discussed here along with some examples of the beneficial effects of these new therapeutic agents. PMID:25165560

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

    PubMed

    Gordon, S G; Mourad, A M

    1991-12-01

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

  6. Passive Treatment And Monitoring At The Standard Mine Superfund Site, Crested Butte, CO (Presentation)

    EPA Science Inventory

    At the 2008 ASMR conference, data from the initial two months of operation of a U.S. EPA pilot biochemical reactor (BCR) was reported. The BCR was designed and constructed in August, 2007 to treat mining influenced water (MIW) emanating from an adit at a remote site in southern ...

  7. Chemical, physical, and biological properties of compounds present at hazardous-waste sites. Final report

    SciTech Connect

    Not Available

    1985-09-27

    The chemical profiles are intended to serve as a concise reference with information on the physicochemical properties, transport and fate, toxicity, and regulatory standards for individual chemicals identified by the EPA Office of Waste Program Enforcement at hazardous-waste sites. The profiles can be used in conjunction with the Toxicology and Endangerment Assessment Handbooks.

  8. Nuclear Site Security in the Event of Terrorist Activity

    SciTech Connect

    Thomson, M.L.; Sims, J.

    2008-07-01

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

  9. BNL Activities in Advanced Neutron Source Development: Past and Present

    SciTech Connect

    Hastings, J.B.; Ludewig, H.; Montanez, P.; Todosow, M.; Smith, G.C.; Larese, J.Z.

    1998-06-14

    Brookhaven National Laboratory has been involved in advanced neutron sources almost from its inception in 1947. These efforts have mainly focused on steady state reactors beginning with the construction of the first research reactor for neutron beams, the Brookhaven Graphite Research Reactor. This was followed by the High Flux Beam Reactor that has served as the design standard for all the subsequent high flux reactors constructed worldwide. In parallel with the reactor developments BNL has focused on the construction and use of high energy proton accelerators. The first machine to operate over 1 GeV in the world was the Cosmotron. The machine that followed this, the AGS, is still operating and is the highest intensity proton machine in the world and has nucleated an international collaboration investigating liquid metal targets for next generation pulsed spallation sources. Early work using the Cosmotron focused on spallation product studies for both light and heavy elements into the several GeV proton energy region. These original studies are still important today. In this report we discuss the facilities and activities at BNL focused on advanced neutron sources. BNL is involved in the proton source for the Spallation Neutron source, spectrometer development at LANSCE, target studies using the AGS and state-of-the-art neutron detector development.

  10. BNL ACTIVITIES IN ADVANCED NEUTRON SOURCE DEVELOPMENT: PAST AND PRESENT

    SciTech Connect

    HASTINGS,J.B.; LUDEWIG,H.; MONTANEZ,P.; TODOSOW,M.; SMITH,G.C.; LARESE,J.Z.

    1998-06-14

    Brookhaven National Laboratory has been involved in advanced neutron sources almost from its inception in 1947. These efforts have mainly focused on steady state reactors beginning with the construction of the first research reactor for neutron beams, the Brookhaven Graphite Research Reactor. This was followed by the High Flux Beam Reactor that has served as the design standard for all the subsequent high flux reactors constructed worldwide. In parallel with the reactor developments BNL has focused on the construction and use of high energy proton accelerators. The first machine to operate over 1 GeV in the world was the Cosmotron. The machine that followed this, the AGS, is still operating and is the highest intensity proton machine in the world and has nucleated an international collaboration investigating liquid metal targets for next generation pulsed spallation sources. Early work using the Cosmotron focused on spallation product studies for both light and heavy elements into the several GeV proton energy region. These original studies are still important today. In the sections below the authors discuss the facilities and activities at BNL focused on advanced neutron sources. BNL is involved in the proton source for the Spallation Neutron source, spectrometer development at LANSCE, target studies using the AGS and state-of-the-art neutron detector development.

  11. Present status of some technological activities supporting the MOLCARE project

    SciTech Connect

    Torazza, A.; Rocchini, G.; Scagliotti, M.

    1996-12-31

    The development of MCFC stack technology is carried out at Ansaldo Ricerche in the framework of the MOLCARE project, a cooperation with Spanish companies under a partial UE funding, while a specific research program concerning the physico-chemical characterization of materials is performed jointly by CISE and ENEL. The project includes the development, the construction and the testing of a full scale 100 kW prototype, the assessment of stack technology on subscale stacks, the mathematical modelling of the MCFC based plants and the basic researches. The aim of the basic researches, carried out on single cells, is to improve the effectiveness and durability of both the active and the hardware materials. The Ansaldo stack technology is based on external manifolding. The full scale 100 kW prototype will be integrated with the sensible heat reformer and other ancillary equipments according to the {open_quote}Compact Unit (CU){close_quotes} concept. These technical choices stress requirements for manifold gasket configuration. electrolyte migration control, {Delta}p management and porous component compaction.

  12. Forecasting the Peak of the Present Solar Activity Cycle

    NASA Astrophysics Data System (ADS)

    Hamid, Rabab; Marzouk, Beshir

    2016-07-01

    Solar forecasting of the level of sun Activity is very important subject for all space programs. Most predictions are based on the physical conditions prevailing at or before the solar cycle minimum preceding the maximum in question. Our aim is to predict the maximum peak of cycle 24 using precursor techniques in particular those using spotless event, geomagnetic aa min. index and solar flux F10.7. Also prediction of exact date of the maximum (Tr) is taken in consideration. A study of variation over previous spotless event for cycles 7-23 and that for even cycles (8-22) are carried out for the prediction. Linear correlation between RM and spotless event around the preceding minimum gives RM24t = 101.9with rise time Tr = 4.5 Y. For the even cycles RM24e = 108.3 with rise time Tr = 3.9 Y. Based on the average aa min. index for the year of sunspot minimum cycles (13 - 23), we estimate the expected amplitude for cycle 24 to be RMaa = 116.5 for both the total and even cycles. Application of the data of solar flux F10.7 which cover only cycles (19-23) was taken in consideration and gives predicted maximum amplitude R24 10.7 = 146, which are over estimation. Our result indicating a somewhat weaker cycle 24 as compared to cycles 21-23.

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

    SciTech Connect

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

    2012-02-06

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

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

    PubMed

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

    1986-05-01

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

  15. Present-day aeolian activity in Herschel Crater, Mars

    NASA Astrophysics Data System (ADS)

    Cardinale, Marco; Silvestro, Simone; Vaz, David A.; Michaels, Timothy; Bourke, Mary C.; Komatsu, Goro; Marinangeli, Lucia

    2016-02-01

    In this report, we show evidence for ripple and dune migration in Herschel Crater on Mars. We estimate an average dune migration of 0.8 m and a minimum ripple migration of 1.1 m in a time span of 3.7 Earth-years. These dunes and ripples are mainly shaped by prevailing winds coming from the north, however we also report the presence of secondary winds which elongate the barchans' horns. Such a complex wind scenario is likely caused by the influence of winds blowing off the western crater rim as suggested by the Mars Regional Atmospheric Modeling System (MRAMS), an atmospheric mesoscale model. A multi-directional wind regime at the local scale is also supported by the observed bimodal distribution of the ripple trends. For the first time, a survey integrating the assessment of dune and ripple migration is presented, showing how dune topography can influence the migration patterns of ripples and how underlying topography appears to control the rates of dune migration.

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

    USGS Publications Warehouse

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

    2000-01-01

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

  17. The accelerated site technology deployment program presents the segmented gate system

    SciTech Connect

    PATTESON,RAYMOND; MAYNOR,DOUG; CALLAN,CONNIE

    2000-02-24

    The Department of Energy (DOE) is working to accelerate the acceptance and application of innovative technologies that improve the way the nation manages its environmental remediation problems. The DOE Office of Science and Technology established the Accelerated Site Technology Deployment Program (ASTD) to help accelerate the acceptance and implementation of new and innovative soil and ground water remediation technologies. Coordinated by the Department of Energy's Idaho Office, the ASTD Program reduces many of the classic barriers to the deployment of new technologies by involving government, industry, and regulatory agencies in the assessment, implementation, and validation of innovative technologies. The paper uses the example of the Segmented Gate System (SGS) to illustrate how the ASTD program works. The SGS was used to cost effectively separate clean and contaminated soil for four different radionuclides: plutonium, uranium, thorium, and cesium. Based on those results, it has been proposed to use the SGS at seven other DOE sites across the country.

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

    SciTech Connect

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

    2014-08-14

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

  19. The Three Mycobacterium tuberculosis Antigen 85 Isoforms Have Unique Substrates and Activities Determined by Non-active Site Regions*

    PubMed Central

    Backus, Keriann M.; Dolan, Michael A.; Barry, Conor S.; Joe, Maju; McPhie, Peter; Boshoff, Helena I. M.; Lowary, Todd L.; Davis, Benjamin G.; Barry, Clifton E.

    2014-01-01

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro216–Phe228 loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors. PMID:25028517

  20. Alternative autogenous bone graft donor sites in brachymetatarsia reconstruction: a review of the literature with clinical presentations.

    PubMed

    Kashuk, K B; Hanft, J R; Schabler, J A; Kopelman, J

    1991-01-01

    The authors present a literature review on the surgical treatment of brachymetatarsia. They discuss the identification of donor sites for autogenous bone graft harvesting from the foot. Three case reports of brachymetatarsia, one of iatrogenic, and two of congenital origin are presented. PMID:1874999

  1. Assessment of present day geomorphological dynamics to decipher landscape evolution around the Paleolithic sites of Melka Kunture, Ethiopia

    NASA Astrophysics Data System (ADS)

    Maerker, Michael; Schillaci, Calogero; Melis, Rita; Mussi, Margherita

    2014-05-01

    The area of Melka Kunture (central Ethiopia) is one of the most important clusters of Paleolithic sites in Eastern Africa. The archaeological record spans from c. 1.7 Ma onwards, with a number of stratified occurrences of Oldowan, Acheulean, Middle Stone Age and Late Stone Age industries, together with faunal remains and human fossils. However, the archaeological sites are endangered by flooding and soil erosion. The main excavation area lies close to the convergence of the Awash river with the Atabella river, one of the main tributaries of the upper Awash catchment. In the semi-arid Ethiopian highlands, gully networks develop especially in the vicinity of the active and inactive river meanders. Various erosion processes are linked to specific driving factors such as the rainfall regime, the land use/cover changes and vertic soils with a specific hydrological behaviour. It was documented in the field and by previous research that the origin of most of the man made erosion channels is due to animal pathways and car tracks. However, paleolandscape features increase the general erosion risk. Former wetland areas and deposition zones are particularly affected by soil erosion processes. Hence, the spatial distribution and characteristics of present day geomorphic processes also reveal information on the paleolandscape. In order to assess landscape evolution and present day geomorphologic dynamics, we mapped the geomorphology describing in detail the present-day slope processes at a 10.000 scale. We performed a detailed terrain analysis based on high resolution DEMs such as SRTM-X with 25m resolution and ALOS/PRISM with 10m resolution to characterize the main erosion processes and surface runoff dynamics. The latter ones are simulated using a Soil Conservation Service Curve Number method. Landuse was delineated for a larger area using ASTER 25m multispectral data. Finally, using calibrated topographic indices and a simple hydrological model we were able to detect and

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

    SciTech Connect

    Ashby, J.; Rowe, J.

    1980-02-01

    The primary consideration for the suitability of a nuclear waste repository site is the overall ability of the repository to safely contain radioactive waste. This report is a discussion of the past, present, and future effects of resource activities on waste containment. Past and present resource activities which provide release pathways (i.e., leaky boreholes, adjacent mines) will receive initial evaluation during the early stages of any repository site study. However, other resource activities which may have subtle effects on containment (e.g., long-term pumping causing increased groundwater gradients, invasion of saline water causing lower retardation) and all potential future resource activities must also be considered during the site evaluation process. Resource activities will affect both the siting and the designing of repositories. Ideally, sites should be located in areas of low resource activity and low potential for future activity, and repository design should seek to eliminate or minimize the adverse effects of any resource activity. Buffer zones should be created to provide areas in which resource activities that might adversely affect containment can be restricted or curtailed. This could mean removing large areas of land from resource development. The impact of these frozen assets should be assessed in terms of their economic value and of their effect upon resource reserves. This step could require a major effort in data acquisition and analysis followed by extensive numerical modeling of regional fluid flow and mass transport. Numerical models should be used to assess the effects of resource activity upon containment and should include the cumulative effects of different resource activities. Analysis by other methods is probably not possible except for relatively simple cases.

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

    PubMed

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

    1988-07-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. Tritium processing at the Savannah River Site (SRS): Present and future

    SciTech Connect

    Ortman, M.S.; Heung, L.K.; Nobile, A.; Rabun, R.L. III.

    1989-01-01

    Tritium handling equipment and methods at the Savannah River Site Tritium Facilities have been continually improved since tritium processing operations began in 1955. Several new technologies were introduced into the Tritium Facilities in the 1980's. One of these is the use of fluidless, mechanical pumps (Normetex and Metal Bellows) to replace mercury pumps. A second is the use of metal hydride technology to store, purify, isotopically separate, pump, and compress hydrogen isotopes. Metal hydrides, such as La-Ni-Al alloys and Pd loaded on kieselguhr, offer significant flexibility and size advantages compared with conventional tritium handling technology, such as gas tanks, thermal diffusion columns, and mechanical compressors. Metal hydrides have been used in the Tritium Facilities since 1984 with the most important application of this technology being planned for the Replacement Tritium Facility, a $140 million facility scheduled for completion in 1990 and startup in 1991. 11 refs., 9 figs.

  6. Development of Alkaline Oxidative Dissolution Methods for Chromium (III) Compounds Present in Hanford Site Tank Sludges

    SciTech Connect

    NN Krot; VP Shilov; AM Fedoseev; NA Budantseva; MV Nikonov; AB Yusov; AYu Garnov; IA Charushnikova; VP Perminov; LN Astafurova; TS Lapitskaya; VI Makarenkov

    1999-07-02

    The high-level radioactive waste sludge in the underground storage tanks at the Hanford Site contains various chromium(III)solid phases. Dissolution and removal of chromium from tank waste sludges is desirable prior to high-level waste vitrification because increased volume is required to incorporate the residual chromium. Unfortunately, dissolution of chromium from the sludge to form Cr(OH){sub 4}{sup {minus}} through treatment with heated NaOH solution (also used to dissolve aluminum phases and metathesize phosphates to sodium salts) generally has been unsuccessful in tests with both simulated and genuine Hanford waste sludges. Oxidative dissolution of the Cr(III) compounds to form soluble chromate has been proposed as an alternative chromium solid phase dissolution method and results of limited prior testing have been reported.

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

    SciTech Connect

    1981-02-27

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

  8. An online support site for preparation of oral presentations in science and engineering

    NASA Astrophysics Data System (ADS)

    Kunioshi, Nílson; Noguchi, Judy; Hayashi, Hiroko; Tojo, Kazuko

    2012-12-01

    Oral communication skills are essential for engineers today and, as they are included in accreditation criteria of educational programmes, their teaching and evaluation deserve attention. However, concrete aspects as to what should be taught and evaluated in relation to oral communication skills have not been sufficiently established. In this paper, a method to aid the efficient teaching of oral presentation skills is proposed, from the presentation structure level to word and sentence level choices, through the use of JECPRESE, The Japanese-English Corpus of Presentations in Science and Engineering. As of June 2012, the corpus is composed of transcriptions of 74 presentations delivered in Japanese by students graduating from the Master's programme of various engineering departments and 31 presentations delivered in English, 16 by experienced researchers at an international conference on chemistry, and 15 by undergraduate engineering students of a mid-sized American university. The utterances were classified according to the specific moves (sections of the speech that express specific speaker intent) appearing in the presentations and frequently used words/expressions to express these moves were identified.

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

    PubMed

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

    2015-04-21

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

  10. Strategies and Activities for Using Local Communities as Environmental Education Sites.

    ERIC Educational Resources Information Center

    Roth, Charles E.; Lockwood, Linda G.

    Presented are over 100 environmental education activities which use the local community for a learning site and resource. These lessons are grouped under seven topical headings: (1) biological neighbors, (2) physical environs, (3) built environs, (4) social environs, (5) understanding ourselves, (6) influencing change, and (7) improvement and…

  11. 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

  12. Electrostatic fields in the active sites of lysozymes.

    PubMed

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

    1989-07-01

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

  13. Histidine at the active site of Neurospora tyrosinase.

    PubMed

    Pfiffner, E; Lerch, K

    1981-10-13

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

  14. Modeling adsorption rate of organic micropollutants present in landfill leachates onto granular activated carbon.

    PubMed

    Ocampo-Pérez, Raúl; Abdel daiem, Mahmoud M; Rivera-Utrilla, José; Méndez-Díaz, José D; Sánchez-Polo, Manuel

    2012-11-01

    The overall adsorption rate of single micropollutants present in landfill leachates such as phthalic acid (PA), bisphenol A (BPA), diphenolic acid (DPA), 2,4-dichlorophenoxy-acetic acid (2,4-D), and 4-chloro-2-methylphenoxyacetic acid (MCPA) on two commercial activated carbons was studied. The experimental data obtained were interpreted by using a diffusional model (PVSDM) that considers external mass transport, intraparticle diffusion, and adsorption on an active site. Furthermore, the concentration decay data were interpreted by using kinetics models. Results revealed that PVSDM model satisfactorily fitted the experimental data of adsorption rate on activated carbon. The tortuosity factor of the activated carbons used ranged from 2 to 4. The contribution of pore volume diffusion represented more than 92% of intraparticle diffusion confirming that pore volume diffusion is the controlling mechanism of the overall rate of adsorption and surface diffusion can be neglected. The experimental data were satisfactorily fitted the kinetic models. The second-order kinetic model was better fitted the experimental adsorption data than the first-order model. PMID:22858399

  15. Geochemistry and geobiology of a present-day serpentinization site in California: The Cedars

    NASA Astrophysics Data System (ADS)

    Morrill, Penny L.; Kuenen, J. Gijs; Johnson, Orion J.; Suzuki, Shino; Rietze, Amanda; Sessions, Alex L.; Fogel, Marilyn L.; Nealson, Kenneth H.

    2013-05-01

    Ultra-basic (pH 11-12) reducing (-656 to -585 mV) groundwater springs discharging from serpentinized peridotite of The Cedars, CA, were investigated for their geochemistry and geobiology. The spring waters investigated were of meteoric origin; however, geochemical modeling suggests that there were two sources of groundwater, a shallow source with sufficient contact with The Cedars' peridotite body to be altered geochemically by serpentinization, and a deeper groundwater source that not only flows through the peridotite body but was also in contact with the marine sediments of the Franciscan Subduction Complex (FSC) below the peridotite body. We propose that the groundwater discharging from lower elevations (GPS1 and CS1) reflect the geochemistry of the deeper groundwater in contact with FSC, while groundwaters discharging from springs at higher elevations (NS1 and BSC) were a mixture of the shallow peridotite-only groundwater and the deeper groundwater that has been in contact with the FSC. Cell densities of suspended microbes within these waters were extremely low. In the NS1 and BSC spring fluids, cell densities ranged from 102 to 103 cells/ml, while suspended cells at GPS were lower than 10 cells/mL. However, glass slides incubated in the BSC and GPS1 springs for 2-3 weeks were colonized by cells with densities ranging from 106 to 107 cells/cm2 attached to their surfaces. All of the springs were very low (⩽1 μM) in several essential elements and electron acceptors (e.g. nitrate/ammonium, sulfate, and phosphate) required for (microbial) growth, which is not uncommon at sites of continental serpentinization. Gases rich in N2, H2, and CH4 were exsolving from the springs. The stable carbon isotope value (δ13CCH4 = -68 ± 0.6‰) and the CH4/C2+ (>103) of methane and other gaseous hydrocarbons exsolving from NS1 were typical of microbially sourced methane, whereas the isotope values and the CH4/C2+ of BSC and CS1 springs were more enriched in 13C and had CH4/C2

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

    PubMed

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

    1995-02-28

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

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

    SciTech Connect

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

    1995-12-31

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

  18. The copper active site of CBM33 polysaccharide oxygenases.

    PubMed

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

    2013-04-24

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

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

    Nosrati, Geoffrey R; Houk, K N

    2012-01-01

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

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

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-05-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2004-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    SciTech Connect

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

    1991-09-10

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

  7. Benign Eccrine Tumors Presenting on the Penis: Common Tumors at an Uncommon Site.

    PubMed

    Tan, Aaron Wei Min; Koh, Mark Jean Ann; Ng, See Ket

    2015-01-01

    Case 1 A 60-year-old Chinese man presented to the dermatology clinic with an asymptomatic, 0.5 × 0.5-cm erythematous papule on the penile shaft for a 2-year duration (Figure 1a). His medical problems consisted only of well-controlled hypothyroidism and hypertension. The initial clinical diagnoses considered were pyogenic granuloma or condyloma accuminatum. The excisional biopsy revealed a polypoidal lesion entirely covered by epidermis, with a proliferation of cuboidal, basaloid cells, consistent with an eccrine poroma (Figure 1b). Case 2 A 28-year-old Chinese man presented with an asymptomatic fusiform, 1.4 × 0.4-cm nodule over the ventral aspect of the prepuce, for a 10-year duration (Figure 2a). His medical history was otherwise unremarkable. Lymphangioma was the initial clinical diagnosis but biopsy results revealed large collapsed cystic spaces within the superficial dermis lined by a double layer of epithelial cells with eosinophilic cytoplasm, without evidence of decapitation secretion, consistent with an eccrine hidrocystoma (Figure 2b). PMID:26861438

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

    PubMed

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

    2016-04-22

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

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

    SciTech Connect

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

    1986-03-01

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

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

    PubMed

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

    2016-04-22

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

  11. P2X7 Receptor Activation Impairs Exogenous MHC Class I Oligopeptides Presentation in Antigen Presenting Cells

    PubMed Central

    Baroja-Mazo, Alberto; Barberà-Cremades, Maria; Pelegrín, Pablo

    2013-01-01

    Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8+ T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5′-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8+ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8+ T cell immunity. PMID:23940597

  12. P2X7 receptor activation impairs exogenous MHC class I oligopeptides presentation in antigen presenting cells.

    PubMed

    Baroja-Mazo, Alberto; Barberà-Cremades, Maria; Pelegrín, Pablo

    2013-01-01

    Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8(+) T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5'-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8(+) T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8(+) T cell immunity.

  13. 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

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

    SciTech Connect

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

    2008-04-02

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

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

    SciTech Connect

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

    1992-02-01

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

  16. Comments on Presentation on Industrial Nuclear Explosion Sites in the Russian Federation: Recovery and Institutional Monitoring Problems

    SciTech Connect

    Bradley, Donald J.

    2009-01-01

    The U.S. National Academy of Sciences selected 6 U.S. scientists to review papers prepared by Russian specialists in 6 specific areas of radioactive waste management concern. As one of the U.S. specialists selected, Don Bradley attended a meeting in Moscow, Russia where the papers were formally presented. Following the presentation, eah one was critiqued by the U.S. specialist. In Mr. Bradley's case the topic was contamination at Peaceful Nuclear Explosion test sites (PNE's). The formal title of the meeting was: "Cleaning Up Sites Contaminated with Radioactive Materials". Following discussions with the U.S. team, each of the U.S. specialists was charged with writing up a short comment paper for the U.S. Academy of Sciences. This is Mr. Bradley's comments on the presentation by Kasatkin V.V., Kamnev Ye.N. and Ilyichev V.A. (Rosatom, FGUP VNIPIpromtechnologii) .

  17. Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site.

    PubMed

    Gajadeera, Chathurada S; Zhang, Xinyi; Wei, Yinan; Tsodikov, Oleg V

    2015-02-01

    Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg(2+)-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn(2+)-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn(2+) at 2.1Å resolution. The active site contains two catalytic Mn(2+) binding sites, each half-occupied, reconciling the previously observed 1:1 Mn(2+):enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295-298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme. PMID:25576794

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

    SciTech Connect

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

    1988-10-18

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-03-23

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

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

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

    2014-01-01

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

  7. NMR structure of the active conformation of the Varkud satellite ribozyme cleavage site

    PubMed Central

    Hoffmann, Bernd; Mitchell, G. Thomas; Gendron, Patrick; Major, François; Andersen, Angela A.; Collins, Richard A.; Legault, Pascale

    2003-01-01

    Substrate cleavage by the Neurospora Varkud satellite (VS) ribozyme involves a structural change in the stem-loop I substrate from an inactive to an active conformation. We have determined the NMR solution structure of a mutant stem-loop I that mimics the active conformation of the cleavage site internal loop. This structure shares many similarities, but also significant differences, with the previously determined structures of the inactive internal loop. The active internal loop displays different base-pairing interactions and forms a novel RNA fold composed exclusively of sheared G-A base pairs. From chemical-shift mapping we identified two Mg2+ binding sites in the active internal loop. One of the Mg2+ binding sites forms in the active but not the inactive conformation of the internal loop and is likely important for catalysis. Using the structure comparison program mc-search, we identified the active internal loop fold in other RNA structures. In Thermus thermophilus 16S rRNA, this RNA fold is directly involved in a long-range tertiary interaction. An analogous tertiary interaction may form between the active internal loop of the substrate and the catalytic domain of the VS ribozyme. The combination of NMR and bioinformatic approaches presented here has identified a novel RNA fold and provides insights into the structural basis of catalytic function in the Neurospora VS ribozyme. PMID:12782785

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

    PubMed Central

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

    2006-01-01

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

  9. CO Oxidation on Au/TiO2: Condition-Dependent Active Sites and Mechanistic Pathways.

    PubMed

    Wang, Yang-Gang; Cantu, David C; Lee, Mal-Soon; Li, Jun; Glezakou, Vassiliki-Alexandra; Rousseau, Roger

    2016-08-24

    We present results of ab initio electronic structure and molecular dynamics simulations (AIMD), as well as a microkinetic model of CO oxidation catalyzed by TiO2 supported Au nanocatalysts. A coverage-dependent microkinetic analysis, based on energetics obtained with density functional methods, shows that the dominant kinetic pathway, activated oxygen species, and catalytic active sites are all strongly depended on both temperature and oxygen partial pressure. Under oxidizing conditions and T < 400 K, the prevalent pathway involves a dynamic single atom catalytic mechanism. This reaction is catalyzed by a transient Au-CO species that migrates from the Au-cluster onto a surface oxygen adatom. It subsequently reacts with the TiO2 support via a Mars van Krevelen mechanism to form CO2 and finally the Au atom reintegrates back into the gold cluster to complete the catalytic cycle. At 300 ≤ T ≤ 600 K, oxygen-bound single Oad-Au(+)-CO sites and the perimeter Au-sites of the nanoparticle work in tandem to optimally catalyze the reaction. Above 600 K, a variety of alternate pathways associated with both single-atom and the perimeter sites of the Au nanoparticle are found to be active. Under low oxygen pressures, Oad-Au(+)-CO species can be a source of catalyst deactivation and the dominant pathway involves only Au-perimeter sites. A detailed comparison of the current model and the existing literature resolves many apparent inconsistencies in the mechanistic interpretations. PMID:27480512

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

    PubMed

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

    1995-10-01

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

  11. Active Site Detection by Spatial Conformity and Electrostatic Analysis—Unravelling a Proteolytic Function in Shrimp Alkaline Phosphatase

    PubMed Central

    Chakraborty, Sandeep; Minda, Renu; Salaye, Lipika; Bhattacharjee, Swapan K.; Rao, Basuthkar J.

    2011-01-01

    Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - CataLytic Active Site Prediction (CLASP). In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP), where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in vitro. PMID

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

    PubMed

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

    2015-02-24

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

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

    SciTech Connect

    Carlton, W.H.; Denham, M.

    1996-07-01

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

  14. Application of Crunch-Flow Routines to Constrain Present and Past Carbon Fluxes at Gas-Hydrate Bearing Sites

    SciTech Connect

    Torres, Marta

    2014-01-31

    In November 2012, Oregon State University initiated the project entitled: Application of Crunch-Flow routines to constrain present and past carbon fluxes at gas-hydrate bearing sites. Within this project we developed Crunch-Flow based modeling modules that include important biogeochemical processes that need to be considered in gas hydrate environments. Our modules were applied to quantify carbon cycling in present and past systems, using data collected during several DOE-supported drilling expeditions, which include the Cascadia margin in US, Ulleung Basin in South Korea, and several sites drilled offshore India on the Bay of Bengal and Andaman Sea. Specifically, we completed modeling efforts that: 1) Reproduce the compositional and isotopic profiles observed at the eight drilled sites in the Ulleung Basin that constrain and contrast the carbon cycling pathways at chimney (high methane flux) and non-chimney sites (low methane, advective systems); 2) Simulate the Ba record in the sediments to quantify the past dynamics of methane flux in the southern Hydrate Ridge, Cascadia margin; and 3) Provide quantitative estimates of the thickness of individual mass transport deposits (MTDs), time elapsed after the MTD event, rate of sulfate reduction in the MTD, and time required to reach a new steady state at several sites drilled in the Krishna-Godavari (K-G) Basin off India. In addition we developed a hybrid model scheme by coupling a home-made MATLAB code with CrunchFlow to address the methane transport and chloride enrichment at the Ulleung Basins chimney sites, and contributed the modeling component to a study focusing on pore-scale controls on gas hydrate distribution in sediments from the Andaman Sea. These efforts resulted in two manuscripts currently under review, and contributed the modeling component of another pare, also under review. Lessons learned from these efforts are the basis of a mini-workshop to be held at Oregon State University (Feb 2014) to instruct

  15. Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation

    PubMed Central

    Lee, Tai-Sung; Giambaşu, George M.; Sosa, Carlos P.; Martick, Monika; Scott, William G.; York, Darrin M.

    2009-01-01

    The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in the hammerhead ribozyme has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence or absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the hammerhead ribozyme folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of hammerhead ribozyme catalysis and support the supposition that Mg2+, in addition to stabilizing active conformations, plays a specific chemical role in catalysis. PMID:19265710

  16. Electron tunnelling through azurin is mediated by the active site Cu ion

    NASA Astrophysics Data System (ADS)

    Alessandrini, Andrea; Gerunda, Mimmo; Canters, G. W.; Verbeet, M. Ph.; Facci, Paolo

    2003-07-01

    Cu- and Zn-azurin chemisorbed on Au(1 1 1) have been comparatively investigated by electrochemical scanning tunnelling microscopy in buffer solution. Cu-azurin shows a marked tunnelling current resonance upon substrate potential at -0.21 V (vs SCE), whereas Zn counterparts do not. These data, discussed in terms of current theories on electron tunnelling through redox adsorbates, demonstrate the role of the electroactive metal ion present in the active site in assisting electron transfer via this metalloprotein.

  17. Spatiotemporally synchronized cancer combination therapy using photo-activated nanoparticle drug delivery systems (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Hasan, Tayyaba

    2016-03-01

    This talk will introduce a new nanotechnology platform for cancer combination therapy that utilizes near infrared light activation not only for photodynamic damage but also as an extrinsic mechanism to initiate release of complimentary drugs to suppress dynamic bursts in molecular signaling networks that promote tumor cell survival and treatment escape. The goal is to achieve co-delivery with concomitant activity of photodynamic, molecular inhibitor and chemotherapeutic agents, selectively within the tumor. This approach overcomes challenges in achieving synergistic interactions using sequential drug delivery. Conventional drug delivery is compromised by the differential pharmacokinetics of individual agents and potentially antagonistic effects—such as vascular shutdown by one agent that limits delivery of the second. Here, photodynamic damage—which efficiently kills drug-resistant cells via damage of common proteins involved in drug-resistance (such as anti-apoptosis factors and drug-efflux transporters)—is synchronized spatially and temporally with the photo-initiated release of complimentary agents—to enable full interaction amongst the individual therapies. This spatiotemporal synchronization offers new prospects for exploiting time-sensitive synergistic interactions. Specific implementations of these concepts will be presented in preclinical models of cancer. Strategies to enable molecular-targeting of cancer cells via site-specific attachment of targeting moieties to the outer lipid shell of these nanovehicles will also be discussed. If successful in humans, this new paradigm for synchronized, tumor-focused combination therapy will ultimately supersede the present use of chronic drug injection by increasing efficacy per cycle whilst reducing systemic exposure to toxic drugs.

  18. 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.

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

    PubMed Central

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

    1999-01-01

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

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

    SciTech Connect

    Hall, K.S.

    1986-01-01

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

  1. Detection limit for activation measurements in ultralow background sites

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2014-06-01

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

  5. A Frontier Molecular Orbital determination of the active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p and d orbital energy levels of the different types of surface sites present on a dispersed metal catalysts. The basis for these calculations is the reported finding that a large number of catalyzed reactions take place on single atom active sites on the metal surface. Thus, these sites can be considered as surface complexes made up of the central active atom surrounded by near-neighbor metal atom ``ligands`` with localized surface orbitals perturbed only by these ``ligands``. These ``complexes`` are based on a twelve coordinate species with the ``ligands`` attached to the t{sub 2g} orbitals and the coordinate axes coincident with the direction of the e{sub g} orbitals on the central atom. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  6. A Frontier Molecular Orbital determination of the active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p and d orbital energy levels of the different types of surface sites present on a dispersed metal catalysts. The basis for these calculations is the reported finding that a large number of catalyzed reactions take place on single atom active sites on the metal surface. Thus, these sites can be considered as surface complexes made up of the central active atom surrounded by near-neighbor metal atom ligands'' with localized surface orbitals perturbed only by these ligands''. These complexes'' are based on a twelve coordinate species with the ligands'' attached to the t{sub 2g} orbitals and the coordinate axes coincident with the direction of the e{sub g} orbitals on the central atom. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  7. The ribotoxin restrictocin recognizes its RNA substrate by selective engagement of active site residues.

    PubMed

    Plantinga, Matthew J; Korennykh, Alexei V; Piccirilli, Joseph A; Correll, Carl C

    2011-04-12

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

  8. Targeting Large Kinase Active Site with Rigid, Bulky Octahedral Ruthenium Complexes

    SciTech Connect

    Maksimoska, Jasna; Feng, Li; Harms, Klaus; Yi, Chunling; Kissil, Joseph; Marmorstein, Ronen; Meggers, Eric

    2009-09-02

    A strategy for targeting protein kinases with large ATP-binding sites by using bulky and rigid octahedral ruthenium complexes as structural scaffolds is presented. A highly potent and selective GSK3 and Pim1 half-sandwich complex NP309 was successfully converted into a PAK1 inhibitor by making use of the large octahedral compounds {Lambda}-FL172 and {Lambda}-FL411 in which the cyclopentadienyl moiety of NP309 is replaced by a chloride and sterically demanding diimine ligands. A 1.65 {angstrom}cocrystal structure of PAK1 with {Lambda}-FL172 reveals how the large coordination sphere of the ruthenium complex matches the size of the active site and serves as a yardstick to discriminate between otherwise closely related binding sites.

  9. Targeted identification of infection-related HLA class I-presented epitopes by stable isotope tagging of epitopes (SITE).

    PubMed

    Meiring, H D; Soethout, E C; de Jong, A P J M; van Els, C A C M

    2007-05-01

    Identification of peptides presented in human leukocyte antigen (HLA) class I molecules after viral infection is of strategic importance for immunology and vaccine development. A powerful strategy aimed at the rapid, unambiguous identification of naturally processed HLA class I-associated peptides, which are induced by viral infection, is presented here. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues for the human leukocyte antigen allele of interest. Subsequently, these cells are mixed with an equal number of noninfected cells, which are cultured in normal medium. Finally, peptides are acid-eluted from immunoprecipitated HLA molecules and subjected to two-dimensional nanoscale liquid chromatography-mass spectrometry analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules.

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

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

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

    Code of Federal Regulations, 2014 CFR

    2014-01-01

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

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

    Code of Federal Regulations, 2013 CFR

    2013-01-01

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

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

    Code of Federal Regulations, 2011 CFR

    2011-01-01

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

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

    Code of Federal Regulations, 2012 CFR

    2012-01-01

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

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

    PubMed

    Hörning, Marcel; Takagi, Seiji; Yoshikawa, Kenichi

    2012-06-01

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

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

    NASA Astrophysics Data System (ADS)

    Hörning, Marcel; Takagi, Seiji; Yoshikawa, Kenichi

    2012-06-01

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

  17. Synthesis of supported bimetallic nanoparticles with controlled size and composition distributions for active site elucidation

    SciTech Connect

    Hakim, Sikander H.; Sener, Canan; Alba Rubio, Ana C.; Gostanian, Thomas M.; O'neill, Brandon J; Ribeiro, Fabio H.; Miller, Jeffrey T.; Dumesic, James A

    2015-08-01

    Elucidation of active sites in supported bimetallic catalysts is complicated by the high level of dispersity in the nanoparticle size and composition that is inherent in conventional methods of catalyst preparation. We present a synthesis strategy that leads to highly dispersed, bimetallic nanoparticles with uniform particle size and composition by means of controlled surface reactions. We demonstrate the synthesis of three systems, RhMo, PtMo, and RhRe, consisting of a highly reducible metal with an oxophilic promoter. These catalysts are characterized by FTIR, CO chemisorption, STEM/EDS, TPR, and XAS analysis. The catalytic properties of these bimetallic nanoparticles were probed for the selective CO hydrogenolysis of (hydroxymethyl)tetrahydropyran to produce 1,6 hexanediol. Based on the characterization results and reactivity trends, the active sites in the hydrogenolysis reaction are identified to be small ensembles of the more noble metal (Rh, Pt) adjacent to highly reduced moieties of the more oxophilic metal (Mo, Re).

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

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

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

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

    SciTech Connect

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

    1993-12-01

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

  20. Dynamically achieved active site precision in enzyme catalysis.

    PubMed

    Klinman, Judith P

    2015-02-17

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

  1. 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.

  2. Site-dependent catalytic activity of graphene oxides towards oxidative dehydrogenation of propane.

    PubMed

    Tang, Shaobin; Cao, Zexing

    2012-12-28

    Graphene oxides (GOs) may offer extraordinary potential in the design of novel catalytic systems due to the presence of various oxygen functional groups and their unique electronic and structural properties. Using first-principles calculations, we explore the plausible mechanisms for the oxidative dehydrogenation (ODH) of propane to propene by GOs and the diffusion of the surface oxygen-containing groups under an external electric field. The present results show that GOs with modified oxygen-containing groups may afford high catalytic activity for the ODH of propane to propene. The presence of hydroxyl groups around the active sites provided by epoxides can remarkably enhance the C-H bond activation of propane and the activity enhancement exhibits strong site dependence. The sites of oxygen functional groups on the GO surface can be easily tuned by the diffusion of these groups under an external electric field, which increases the reactivity of GOs towards ODH of propane. The chemically modified GOs are thus quite promising in the design of metal-free catalysis. PMID:22801590

  3. Physical Activity and Pregnancy: Past and Present Evidence and Future Recommendations

    ERIC Educational Resources Information Center

    Symons Downs, Danielle; Chasan-Taber, Lisa; Evenson, Kelly R.; Leiferman, Jenn; Yeo, SeonAe

    2012-01-01

    Purpose: In this review, we provide researchers and practitioners with an overview of the physical activity and pregnancy literature to promote prenatal physical activity, improve measurement, further elucidate the role of activity in reducing maternal health complications, and inform future research. Method: We examined past and present physical…

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed

    Hiyamuta, S; Ito, K

    1994-04-01

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

  6. Men are from Mars, women are from Venus? Examining gender differences in self-presentation on social networking sites.

    PubMed

    Haferkamp, Nina; Eimler, Sabrina C; Papadakis, Anna-Margarita; Kruck, Jana Vanessa

    2012-02-01

    Psychological research on gender differences in self-presentation has already revealed that women place higher priority on creating a positive self-presentation, while men are less concerned about the image they present in face-to-face (ftf) communication. Nowadays, with the extensive use of new media, self-presentation is no longer so closely tied to ftf situations, but can also take place in the online world. Specifically, social networking sites (SNS), such as Facebook or MySpace, offer various features such as profile pictures, groups, and virtual bulletin boards with which users can create elaborated online representations of themselves. What remains open is whether this virtual self-presentation on SNS is subject to gender differences. Based on studies emphasizing gender-related differences in Internet communication and behavior in general, it can be assumed that men and women have different motives regarding their SNS usage as well. A multimethodological study, combining results of an online survey and a content analysis of 106 user profiles, assessed users' diverse motives for participating in SNS in general, and their use of specific profile elements or self-presentation in particular. In this sample of StudiVZ users, women tend to be more likely to use SNS for comparing themselves with others and for searching for information. Men, on the other hand, are more likely to look at other people's profiles to find friends. Moreover, women tend to use group names for their self-presentation and prefer adding portrait photos to their profiles, while men choose full-body shots. PMID:22132897

  7. Men are from Mars, women are from Venus? Examining gender differences in self-presentation on social networking sites.

    PubMed

    Haferkamp, Nina; Eimler, Sabrina C; Papadakis, Anna-Margarita; Kruck, Jana Vanessa

    2012-02-01

    Psychological research on gender differences in self-presentation has already revealed that women place higher priority on creating a positive self-presentation, while men are less concerned about the image they present in face-to-face (ftf) communication. Nowadays, with the extensive use of new media, self-presentation is no longer so closely tied to ftf situations, but can also take place in the online world. Specifically, social networking sites (SNS), such as Facebook or MySpace, offer various features such as profile pictures, groups, and virtual bulletin boards with which users can create elaborated online representations of themselves. What remains open is whether this virtual self-presentation on SNS is subject to gender differences. Based on studies emphasizing gender-related differences in Internet communication and behavior in general, it can be assumed that men and women have different motives regarding their SNS usage as well. A multimethodological study, combining results of an online survey and a content analysis of 106 user profiles, assessed users' diverse motives for participating in SNS in general, and their use of specific profile elements or self-presentation in particular. In this sample of StudiVZ users, women tend to be more likely to use SNS for comparing themselves with others and for searching for information. Men, on the other hand, are more likely to look at other people's profiles to find friends. Moreover, women tend to use group names for their self-presentation and prefer adding portrait photos to their profiles, while men choose full-body shots.

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

    SciTech Connect

    Teese, G.D.

    1995-09-28

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

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

    SciTech Connect

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

    1993-12-01

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

  10. 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.

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

    PubMed

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

    1998-12-30

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

  12. Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco) and Its Active Site for Chemotaxis.

    PubMed

    Dawar, Farman Ullah; Tu, Jiagang; Xiong, Yang; Lan, Jiangfeng; Dong, Xing Xing; Liu, Xiaoling; Khattak, Muhammad Nasir Khan; Mei, Jie; Lin, Li

    2016-01-01

    Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA), a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase) activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS). The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals) at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics. PMID:27589721

  13. Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco) and Its Active Site for Chemotaxis

    PubMed Central

    Dawar, Farman Ullah; Tu, Jiagang; Xiong, Yang; Lan, Jiangfeng; Dong, Xing Xing; Liu, Xiaoling; Khattak, Muhammad Nasir Khan; Mei, Jie; Lin, Li

    2016-01-01

    Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA), a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase) activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS). The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals) at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics. PMID:27589721

  14. Time-efficient docking of flexible ligands into active sites of proteins

    SciTech Connect

    Rarey, M.; Kramer, B.; Lengauer, T.

    1995-12-31

    We present an algorithm for placing flexible molecules in active sites of proteins. The two major goals in the development of our. docking program, called FlexX, are the explicit exploitation of molecular flexibility of the ligand and the development of a model of the docking process that includes the physico-chemical properties of the molecules. The algorithm consists of three phases: The selection of a base fragment, the placement of the base fragment in the active site, and the incremental construction of the ligand inside the active site. Except for the selection of the base fragment, the algorithm runs without manual intervention. The algorithm is tested by reproducing 11 receptor-ligand complexes known from X-ray crystallography. In all cases, the algorithm predicts a placement of the ligand which is similar to the crystal structure (about 1.5 {Angstrom} RMS deviation or less) in a few minutes on a workstation, assuming that the receptor is given in the bound conformation.

  15. Aerosol measurements at a high-elevation site: composition, size, and cloud condensation nuclei activity

    SciTech Connect

    Friedman, Beth; Zelenyuk, Alla; Beranek, Josef; Kulkarni, Gourihar R.; Pekour, Mikhail S.; Hallar, Anna G.; McCubbin, Ian; Thornton, Joel A.; Cziczo, D. J.

    2013-12-09

    We present measurements of CCN concentrations and associated aerosol composition and size properties at a high-elevation research site in March 2011. CCN closure and aerosol hygroscopicity were assessed using simplified assumptions of bulk aerosol properties as well as a new method utilizing single particle composition and size to assess the importance of particle mixing state in CCN activation. Free troposphere analysis found no significant difference between the CCN activity of free tropospheric aerosol and boundary layer aerosol at this location. Closure results indicate that using only size and number information leads to adequate prediction, in the majority of cases within 50%, of CCN concentrations, while incorporating the hygroscopicity parameters of the individual aerosol components measured by single particle mass spectrometry adds to the agreement, in most cases within 20%, between predicted and measured CCN concentrations. For high-elevation continental sites, with largely aged aerosol and low amounts of local area emissions, a lack of chemical knowledge and hygroscopicity may not hinder models in predicting CCN concentrations. At sites influenced by fresh emissions or more heterogeneous particle types, single particle composition information may be more useful in predicting CCN concentrations and understanding the importance of particle mixing state on CCN activation.

  16. Crystal structures of human tissue kallikrein 4: activity modulation by a specific zinc binding site.

    PubMed

    Debela, Mekdes; Magdolen, Viktor; Grimminger, Valerie; Sommerhoff, Christian; Messerschmidt, Albrecht; Huber, Robert; Friedrich, Rainer; Bode, Wolfram; Goettig, Peter

    2006-10-01

    Human tissue kallikrein 4 (hK4) belongs to a 15-member family of closely related serine proteinases. hK4 is predominantly expressed in prostate, activates hK3/PSA, and is up-regulated in prostate and ovarian cancer. We have identified active monomers of recombinant hK4 besides inactive oligomers in solution. hK4 crystallised in the presence of zinc, nickel, and cobalt ions in three crystal forms containing cyclic tetramers and octamers. These structures display a novel metal site between His25 and Glu77 that links the 70-80 loop with the N-terminal segment. Micromolar zinc as present in prostatic fluid inhibits the enzymatic activity of hK4 against fluorogenic substrates. In our measurements, wild-type hK4 exhibited a zinc inhibition constant (IC50) of 16 microM including a permanent residual activity, in contrast to the zinc-independent mutants H25A and E77A. Since the Ile16 N terminus of wild-type hK4 becomes more accessible for acetylating agents in the presence of zinc, we propose that zinc affects the hK4 active site via the salt-bridge formed between the N terminus and Asp194 required for a functional active site. hK4 possesses an unusual 99-loop that creates a groove-like acidic S2 subsite. These findings explain the observed specificity of hK4 for the P1 to P4 substrate residues. Moreover, hK4 shows a negatively charged surface patch, which may represent an exosite for prime-side substrate recognition. PMID:16950394

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2003-12-01

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

  19. Summaries of "You Do It" Activities Presented at the 1978 SASTA Conference in the Riverland.

    ERIC Educational Resources Information Center

    Walsh, Terry

    1980-01-01

    Summarizes "you do it" activities presented at the 1978 South Australian Science Teachers Association Conference. Topics include microscope use and maintenance, chemical slide cells, chemical models, sound waves, microwaves, astronomy, plant keys, reading geological maps, and natural dye processes. (DS)

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

    PubMed

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

    2014-05-21

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

  1. Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop

    PubMed Central

    Camurdanoglu, B. Z.; Hrovat, C.; Dürnberger, G.; Madalinski, M.; Mechtler, K.; Herbst, R.

    2016-01-01

    The neuromuscular junction (NMJ) forms when a motor neuron contacts a muscle fibre. A reciprocal exchange of signals initiates a cascade of signalling events that result in pre- and postsynaptic differentiation. At the centre of these signalling events stands muscle specific kinase (MuSK). MuSK activation, kinase activity and subsequent downstream signalling are crucial for NMJ formation as well as maintenance. Therefore MuSK kinase activity is tightly regulated to ensure proper NMJ development. We have identified a novel serine phosphorylation site at position 751 in MuSK that is increasingly phosphorylated upon agrin stimulation. S751 is also phosphorylated in muscle tissue and its phosphorylation depends on MuSK kinase activity. A phosphomimetic mutant of S751 increases MuSK kinase activity in response to non-saturating agrin concentrations . In addition, basal MuSK and AChR phosphorylation as well as AChR cluster size are increased. We believe that the phosphorylation of S751 provides a novel mechanism to relief the autoinhibition of the MuSK activation loop. Such a lower autoinhibition could foster or stabilize MuSK kinase activation, especially during stages when no or low level of agrin are present. Phosphorylation of S751 might therefore represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMJ maintenance. PMID:27666825

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

    SciTech Connect

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

    1994-09-01

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

  3. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    SciTech Connect

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J.

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  4. Three-dimensional representations of salt-dome margins at four active strategic petroleum reserve sites.

    SciTech Connect

    Rautman, Christopher Arthur; Stein, Joshua S.

    2003-01-01

    Existing paper-based site characterization models of salt domes at the four active U.S. Strategic Petroleum Reserve sites have been converted to digital format and visualized using modern computer software. The four sites are the Bayou Choctaw dome in Iberville Parish, Louisiana; the Big Hill dome in Jefferson County, Texas; the Bryan Mound dome in Brazoria County, Texas; and the West Hackberry dome in Cameron Parish, Louisiana. A new modeling algorithm has been developed to overcome limitations of many standard geological modeling software packages in order to deal with structurally overhanging salt margins that are typical of many salt domes. This algorithm, and the implementing computer program, make use of the existing interpretive modeling conducted manually using professional geological judgement and presented in two dimensions in the original site characterization reports as structure contour maps on the top of salt. The algorithm makes use of concepts of finite-element meshes of general engineering usage. Although the specific implementation of the algorithm described in this report and the resulting output files are tailored to the modeling and visualization software used to construct the figures contained herein, the algorithm itself is generic and other implementations and output formats are possible. The graphical visualizations of the salt domes at the four Strategic Petroleum Reserve sites are believed to be major improvements over the previously available two-dimensional representations of the domes via conventional geologic drawings (cross sections and contour maps). Additionally, the numerical mesh files produced by this modeling activity are available for import into and display by other software routines. The mesh data are not explicitly tabulated in this report; however an electronic version in simple ASCII format is included on a PC-based compact disk.

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

    PubMed Central

    Stec, Boguslaw

    2012-01-01

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

  6. Dynamic formation of single-atom catalytic active sites on ceria-supported gold nanoparticles

    PubMed Central

    Wang, Yang-Gang; Mei, Donghai; Glezakou, Vassiliki-Alexandra; Li, Jun; Rousseau, Roger

    2015-01-01

    Catalysis by gold supported on reducible oxides has been extensively studied, yet issues such as the nature of the catalytic site and the role of the reducible support remain fiercely debated topics. Here we present ab initio molecular dynamics simulations of an unprecedented dynamic single-atom catalytic mechanism for the oxidation of carbon monoxide by ceria-supported gold clusters. The reported dynamic single-atom catalytic mechanism results from the ability of the gold cation to strongly couple with the redox properties of the ceria in a synergistic manner, thereby lowering the energy of redox reactions. The gold cation can break away from the gold nanoparticle to catalyse carbon monoxide oxidation, adjacent to the metal/oxide interface and subsequently reintegrate back into the nanoparticle after the reaction is completed. Our study highlights the importance of the dynamic creation of active sites under reaction conditions and their essential role in catalysis. PMID:25735407

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

    SciTech Connect

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

    1991-10-01

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

  8. Re-Mediating Classroom Activity with a Non-Linear, Multi-Display Presentation Tool

    ERIC Educational Resources Information Center

    Bligh, Brett; Coyle, Do

    2013-01-01

    This paper uses an Activity Theory framework to evaluate the use of a novel, multi-screen, non-linear presentation tool. The Thunder tool allows presenters to manipulate and annotate multiple digital slides and to concurrently display a selection of juxtaposed resources across a wall-sized projection area. Conventional, single screen presentation…

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

    PubMed Central

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

    2010-01-01

    Oligogalacturonate lyases (OGLs; now also classified as pectate lyase family 22) are cytoplasmic enzymes found in pectinolytic members of Enterobacteriaceae, such as the enteropathogen Yersinia enterocolitica. OGLs utilize a β-elimination mechanism to preferentially catalyze the conversion of saturated and unsaturated digalacturonate into monogalacturonate and the 4,5-unsaturated monogalacturonate-like molecule, 5-keto-4-deoxyuronate. To provide mechanistic insights into the specificity of this enzyme activity, we have characterized the OGL from Y. enterocolitica, YeOGL, on oligogalacturonides and determined its three-dimensional x-ray structure to 1.65 Å. The model contains a Mn2+ atom in the active site, which is coordinated by three histidines, one glutamine, and an acetate ion. The acetate mimics the binding of the uronate group of galactourono-configured substrates. These findings, in combination with enzyme kinetics and metal supplementation assays, provide a framework for modeling the active site architecture of OGL. This enzyme appears to contain a histidine for the abstraction of the α-proton in the −1 subsite, a residue that is highly conserved throughout the OGL family and represents a unique catalytic base among pectic active lyases. In addition, we present a hypothesis for an emerging relationship observed between the cellular distribution of pectate lyase folding and the distinct metal coordination chemistries of pectate lyases. PMID:20851883

  10. Parameterization of an Active Thermal Erosion Site, Caribou Creek, Alaska

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

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

    ERIC Educational Resources Information Center

    Heo, Gyeong Mi; Lee, Romee

    2013-01-01

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

  12. Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog

    PubMed Central

    Englert, Markus; Nakamura, Akiyoshi; Wang, Yane-Shih; Eiler, Daniel; Söll, Dieter; Guo, Li-Tao

    2015-01-01

    Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins. PMID:26582921

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

    SciTech Connect

    Not Available

    1993-01-27

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

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

    PubMed

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

    2015-12-29

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

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

    PubMed

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

    2015-03-19

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

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

    PubMed Central

    2015-01-01

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

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

    PubMed

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

    2015-07-13

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

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

    PubMed

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

    2015-07-13

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

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

    PubMed

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

    2016-07-01

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

  20. Insights Into the Effects of Internal Variability, External Variability, and Active Sites on Heterogeneous Ice Nucleation

    NASA Astrophysics Data System (ADS)

    Beydoun, H.; Sullivan, R. C.; Polen, M.

    2015-12-01

    Heterogeneous ice nucleation (HIN) remains one of the outstanding problems in cloud physics and atmospheric science. Experimental challenges in properly simulating HIN processes with relevant atmospheric conditions have largely contributed to the absence of a consistent and comprehensive parameterization. Here we formulate a new ice active surface site-based stochastic model of HIN with the unique feature of invoking a continuum assumption on the ice nucleation activity (contact angle) of an aerosol particle's surface. The result is a particle specific property g that defines a distribution of local surface ice nucleation rates. Upon integration this yields a full freezing probability function for an ice nucleating particle. Current cold plate droplet freezing measurements provide a great resource for studying the freezing ability of many atmospheric aerosol systems. A method based on statistical significance and critical area analysis is presented that can resolve the two-dimensional nature of the ice nucleation ability of aerosol particles: variability in active sites and freezing rates along an individual particle's surface, as well as variability between two particles of the same type in an aerosol population. When applied to published experimental data, the method demonstrates its ability to comprehensively interpret droplet freezing spectra of variable particle mass and surface area concentrations. By fitting the high concentration freezing curves to a statistically significant active site density function, the lower concentration freezing curves are successfully fitted via a process of random sampling from the statistically significant distribution. Using the new scheme, comprehensive parameterizations that can track the frozen fraction of cloud droplets in larger atmospheric models are derived.

  1. Active site studies of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

    SciTech Connect

    Vlahos, C.J.

    1987-01-01

    The data presented delineate the complete amino acid sequence of E. coli KHG aldolase and also identify Lys-133, Glu-45, and Arg-49 as aminoacyl residues required for catalytic activity. Incubation of E. coli KHG aldolase with (/sup 14/C)pyruvate in the presence of NaCNBH/sub 3/ results in the incorporation of one mol of /sup 14/C per mol of enzyme subunit. Digestion of this enzyme-adduct with trypsin, followed by purification of the peptides, allowed for the isolation of a unique radioactive peptide. Its amino acid sequence showed that the pyruvate-binding (i.e., Schiff-base forming) lysine residue is located at position 133 in the intact enzyme. E. coli KHG aldolase activity is lost when the enzyme is reacted with bromopyruvate; saturation kinetics are observed. The substrates, pyruvate and KHG, protect the enzyme from inactivation. Both facts suggest that the reagent is active-site specific. Incubation of the aldolase with (3-/sup 14/C)bromopyruvate is associated with a concomitant loss of enzymatic activity and esterification of Glu-45; if the enzyme is denatured in the presence of excess bromopyruvate, Cys-159 and Cys-180 are also alkylated. Blocking the active-site lysine residue with pyruvate prevents Glu-45 from being esterified but does not eliminate alkylation of these two cysteine residues. Woodward's Reagent K was also found to inactivate the aldolase under conditions that are usually specific for carboxyl group modification. This aldolase is also inactivated by 1,2-cyclohexanedione. Loss of enzymatic activity occurs concomitantly with modification of one arginine residue per enzyme subunit. Treatment of the aldolase with the arginine-specific reagent, 4-(oxyacetyl)phenoxyacetic acid, followed by digestion with trypsin allowed for the isolation of a unique peptide and the identification of Arg-49 as the specific residue involved.

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

    PubMed

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

    2004-10-01

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

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

    PubMed

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

    2015-04-28

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

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

    PubMed Central

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

    2015-01-01

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

  5. NMR structure of the A730 loop of the Neurospora VS ribozyme: insights into the formation of the active site

    PubMed Central

    Bonneau, Eric; Girard, Nicolas; Boisbouvier, Jérôme; Legault, Pascale

    2011-01-01

    The Neurospora VS ribozyme is a small nucleolytic ribozyme with unique primary, secondary and global tertiary structures, which displays mechanistic similarities to the hairpin ribozyme. Here, we determined the high-resolution NMR structure of a stem–loop VI fragment containing the A730 internal loop, which forms part of the active site. In the presence of magnesium ions, the A730 loop adopts a structure that is consistent with existing biochemical data and most likely reflects its conformation in the VS ribozyme prior to docking with the cleavage site internal loop. Interestingly, the A730 loop adopts an S-turn motif that is also present in loop B within the hairpin ribozyme active site. The S-turn appears necessary to expose the Watson–Crick edge of a catalytically important residue (A756) so that it can fulfill its role in catalysis. The A730 loop and the cleavage site loop of the VS ribozyme display structural similarities to internal loops found in the active site of the hairpin ribozyme. These similarities provided a rationale to build a model of the VS ribozyme active site based on the crystal structure of the hairpin ribozyme. PMID:21266483

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

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

    SciTech Connect

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

    2008-11-10

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

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

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

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

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

    PubMed

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

    2006-11-01

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

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

    PubMed

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

    2014-12-12

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

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

    ERIC Educational Resources Information Center

    Pauls, John

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

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

    PubMed

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

    2016-09-12

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

  13. Use of Ground Penetrating Radar and Gradiometry in Identifying Domestic Activity Areas Within the Kolomoki Mounds Archaeological Site, Georgia

    NASA Astrophysics Data System (ADS)

    Serman, N.

    2005-05-01

    The Kolomoki Mounds archaeological site (9ER1) in southwest Georgia appears to be one of the most important Woodland Period (ca. 1000 B.C. - A.D. 900) centers in southeastern United States. The site originally had at least eight mounds, exquisite ceramics and, seemingly, a year-round occupation. Due to an early archaeological misinterpretation, Kolomoki was, until recently, all but ignored in archaeological research. Consequently, the site and its occupation are not well understood. Today, the site is included in the National Register of Historic Places, and is not available for standard archaeological investigation, that is, extensive excavation. Therefore, non-destructive geophysical exploration provides an ideal means for investigating protected sites, such as Kolomoki, to obtain archaeologically relevant information. I will present the results of the first two geophysical surveys I conducted within the Kolomoki Mounds archaeological site in 2001. These surveys are part of my ongoing geophysical research with the purpose of better understanding intra-site settlement patterns at the Kolomoki Mounds archaeological site. The results of the ground-penetrating radar and gradiometry surveys indicate different activity areas at the site. There is a pronounced difference in appearance and density of anomalies between at least two areas, with one of these areas being a part of the habitation area.

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

    PubMed

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

    2013-01-01

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

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

    SciTech Connect

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

    2014-01-03

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

  16. Sediment biogeochemistry and microbial activity at natural hydrocarbon seeps and at sites impacted by anthropogenic hydrocarbon discharges

    NASA Astrophysics Data System (ADS)

    Joye, S. B.; Sibert, R.; Battles, J.; Fields, L.; Kleindienst, S.; Crespo-Medina, M.; Hunter, K.; Meile, C. D.; Montoya, J. P.

    2013-12-01

    , sediment microbial activity from sites near the source of the discharge was distinct from control sites, 10 nm away, and not impacted by the persistent leak. Thus, the microbial communities present at natural hydrocarbon seeps are well adapted to metabolizing the hydrocarbons that persistently flux through the system. However, the microbial communities at anthropogenically-impacted sites exhibit a different response - inhibition of activity - to hydrocarbon addition. Together these data illustrate that anthropogenic hydrocarbon discharges exert clear impacts on the benthos that need to be studied in much more detail.

  17. Earthquake prediction activities and Damavand earthquake precursor test site in Iran

    NASA Astrophysics Data System (ADS)

    Mokhtari, Mohammad

    2010-01-01

    Iran has long been known as one of the most seismically active areas of the world, and it frequently suffers destructive and catastrophic earthquakes that cause heavy loss of human life and widespread damage. The Alborz region in the northern part of Iran is an active EW trending mountain belt of 100 km wide and 600 km long. The Alborz range is bounded by the Talesh Mountains to the west and the Kopet Dagh Mountains to the east and consists of several sedimentary and volcanic layers of Cambrian to Eocene ages that were deformed during the late Cenozoic collision. Several active faults affect the central Alborz. The main active faults are the North Tehran and Mosha faults. The Mosha fault is one of the major active faults in the central Alborz as shown by its strong historical seismicity and its clear morphological signature. Situated in the vicinity of Tehran city, this 150-km-long N100° E trending fault represents an important potential seismic source. For earthquake monitoring and possible future prediction/precursory purposes, a test site has been established in the Alborz mountain region. The proximity to the capital of Iran with its high population density, low frequency but high magnitude earthquake occurrence, and active faults with their historical earthquake events have been considered as the main criteria for this selection. In addition, within the test site, there are hot springs and deep water wells that can be used for physico-chemical and radon gas analysis for earthquake precursory studies. The present activities include magnetic measurements; application of methodology for identification of seismogenic nodes for earthquakes of M ≥ 6.0 in the Alborz region developed by International Institute of Earthquake Prediction Theory and Mathematical Geophysics, IIEPT RAS, Russian Academy of Science, Moscow (IIEPT&MG RAS); a feasibility study using a dense seismic network for identification of future locations of seismic monitoring stations and application

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

    PubMed

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

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

  19. Encroachment of Human Activity on Sea Turtle Nesting Sites

    NASA Astrophysics Data System (ADS)

    Ziskin, D.; Aubrecht, C.; Elvidge, C.; Tuttle, B.; Baugh, K.; Ghosh, T.

    2008-12-01

    The encroachment of anthropogenic lighting on sea turtle nesting sites poses a serious threat to the survival of these animals [Nicholas, 2001]. This danger is quantified by combining two established data sets. The first is the Nighttime Lights data produced by the NOAA National Geophysical Data Center [Elvidge et al., 1997]. The second is the Marine Turtle Database produced by the World Conservation Monitoring Centre (WCMC). The technique used to quantify the threat of encroachment is an adaptation of the method described in Aubrecht et al. [2008], which analyzes the stress on coral reef systems by proximity to nighttime lights near the shore. Nighttime lights near beaches have both a direct impact on turtle reproductive success since they disorient hatchlings when they mistake land-based lights for the sky-lit surf [Lorne and Salmon, 2007] and the lights are also a proxy for other anthropogenic threats. The identification of turtle nesting sites with high rates of encroachment will hopefully steer conservation efforts to mitigate their effects [Witherington, 1999]. Aubrecht, C, CD Elvidge, T Longcore, C Rich, J Safran, A Strong, M Eakin, KE Baugh, BT Tuttle, AT Howard, EH Erwin, 2008, A global inventory of coral reef stressors based on satellite observed nighttime lights, Geocarto International, London, England: Taylor and Francis. In press. Elvidge, CD, KE Baugh, EA Kihn, HW Kroehl, ER Davis, 1997, Mapping City Lights with Nighttime Data from the DMSP Operational Linescan System, Photogrammatic Engineering and Remote Sensing, 63:6, pp. 727-734. Lorne, JK, M Salmon, 2007, Effects of exposure to artificial lighting on orientation of hatchling sea turtles on the beach and in the ocean, Endangered Species Research, Vol. 3: 23-30. Nicholas, M, 2001, Light Pollution and Marine Turtle Hatchlings: The Straw that Breaks the Camel's Back?, George Wright Forum, 18:4, p77-82. Witherington, BE, 1999, Reducing Threats To Nesting Habitat, Research and Management Techniques for

  20. Epidermolytic palmoplantar keratoderma caused by activation of a cryptic splice site in KRT9.

    PubMed

    Fuchs-Telem, D; Padalon-Brauch, G; Sarig, O; Sprecher, E

    2013-03-01

    Epidermolytic palmoplantar keratoderma (EPPK) is caused by mutations in KRT9 and less often, KRT1. All known mutations in KRT9 have been found in regions of the gene encoding the conserved central α-helix rod domain. In the present study, we investigated the molecular basis of EPPK in a patient of Ashkenazi Jewish origin. The patient was found to carry a novel missense mutation in KRT9, resulting in the substitution of a poorly conserved leucine for valine at position 11 of the amino acid sequence. Despite its unusual location, the mutation was shown to be pathogenic through activation of a cryptic donor splice site, resulting in the deletion of 162 amino acids. The present data indicate the need to screen keratin genes in their entirety, as mutations altering domains of lesser functional importance may exert their deleterious effect at the transcriptional level.

  1. Library Services in Transition: A Presentation of Current Activities at the Royal Institute of Technology Library.

    ERIC Educational Resources Information Center

    Schwarz, Stephan, Ed.; And Others

    This collection of 15 essays, reports, and notes written by staff members of the Royal Institute of Technology Library (RITL) addresses various aspects of modern librarianship, and outlines some of the RITL contributions to the field. Designed to present a composite picture of the various activities of RITL since about mid-1973, these essays…

  2. Who Will Present It during the Broadcast? A Case Study at a Daily Activity Centre

    ERIC Educational Resources Information Center

    Reichenberg, Monica

    2016-01-01

    The present study is an investigation of a daily activity centre (DA). The overall aim was to build a grounded theory that could explain why this particular DA deviated from the norms of Swedish group homes and DAs described in previous studies. These studies have suggested that the staff stuck to old routines, such as letting the participants…

  3. Bubble Festival: Presenting Bubble Activities in a Learning Station Format. Teacher's Guide.

    ERIC Educational Resources Information Center

    Barber, Jacqueline; Willard, Carolyn

    This learning station guide adapts the Bubble Festival, an all-school event, for individual classrooms. It presents students with a variety of different challenges at learning stations set up around the classroom. The activities are student-centered and involve open-ended investigations. Also included are ways to extend students' experiences at…

  4. Erythema annulare centrifugum as presenting sign of activation of breast cancer*

    PubMed Central

    Topal, Ilteris Oguz; Topal, Yunus; Sargan, Aytul; Duman, Hatice; Gungor, Sule; Goncu, Ozgur Emek Kocaturk; Ozekinci, Selver

    2015-01-01

    Erythema annulare centrifugum is a figurate erythema of unknown etiology. It has been associated with many different entities, including infections, food allergy, drug reactions and malignant neoplasms. Herein, we report a case of erythema annulare centrifugum as presenting sign of activation of breastcancer. PMID:26734884

  5. Erythema annulare centrifugum as presenting sign of activation of breast cancer.

    PubMed

    Topal, Ilteris Oguz; Topal, Yunus; Sargan, Aytul; Duman, Hatice; Gungor, Sule; Goncu, Ozgur Emek Kocaturk; Ozekinci, Selver

    2015-01-01

    Erythema annulare centrifugum is a figurate erythema of unknown etiology. It has been associated with many different entities, including infections, food allergy, drug reactions and malignant neoplasms. Herein, we report a case of erythema annulare centrifugum as presenting sign of activation of breastcancer.

  6. Stimulation of hormone-responsive adenylate cyclase activity by a factor present in the cell cytosol.

    PubMed Central

    MacNeil, S; Crawford, A; Amirrasooli, H; Johnson, S; Pollock, A; Ollis, C; Tomlinson, S

    1980-01-01

    1. Homogenates of whole tissues were shown to contain both intracellular and extracellular factors that affected particulate adenylate cyclase activity in vitro. Factors present in the extracellular fluids produced an inhibition of basal, hormone- and fluoride-stimulated enzyme activity but factors present in the cell cytosol increased hormone-stimulated activity with relatively little effect on basal or fluoride-stimulated enzyme activity. 2. The existence of this cytosol factor or factors was investigated using freshly isolated human platelets, freshly isolated rat hepatocytes, and cultured cells derived from rat osteogenic sarcoma, rat calvaria, mouse melanoma, pig aortic endothelium, human articular cartilage chondrocytes and human bronchial carcinoma (BEN) cells. 3. The stimulation of the hormone response by the cytosol factor ranged from 60 to 890% depending on the tissue of origin of the adenylate cyclase. 4. In each case the behaviour of the factor was similar to the action of GTP on that particular adenylate cyclase preparation. 5. No evidence of tissue or species specificity was found, as cytosols stimulated adenylate cyclase from their own and unrelated tissues to the same degree. 6. In the human platelet, the inclusion of the cytosol in the assay of adenylate cyclase increased the rate of enzyme activity in response to stimulation by prostaglandin E1 without affecting the amount of prostaglandin E1 required for half-maximal stimulation or the characteristics of enzyme activation by prostaglandin E. PMID:7396869

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

    SciTech Connect

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

    1985-07-02

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

  8. Janus particles as artificial antigen-presenting cells for T cell activation.

    PubMed

    Chen, Bo; Jia, Yilong; Gao, Yuan; Sanchez, Lucero; Anthony, Stephen M; Yu, Yan

    2014-01-01

    Here we show that the multifunctionality of Janus particles can be exploited for in vitro T cell activation. We engineer bifunctional Janus particles on which the spatial distribution of two ligands, anti-CD3 and fibronectin, mimics the "bull's eye" protein pattern formed in the membrane junction between a T cell and an antigen-presenting cell. Different levels of T cell activation can be achieved by simply switching the spatial distribution of the two ligands on the surfaces of the "bull's eye" particles. We find that the ligand pattern also affects clustering of intracellular proteins. This study demonstrates that anisotropic particles, such as Janus particles, can be developed as artificial antigen-presenting cells for modulating T cell activation. PMID:25343426

  9. Identification of a small protein domain present in all plant lineages that confers high prephenate dehydratase activity.

    PubMed

    El-Azaz, Jorge; de la Torre, Fernando; Ávila, Concepción; Cánovas, Francisco M

    2016-07-01

    l-Phenylalanine serves as a building block for the biosynthesis of proteins, but also as a precursor for a wide range of plant-derived compounds essential for plants and animals. Plants can synthesize Phe within the plastids using arogenate as a precursor; however, an alternative pathway using phenylpyruvate as an intermediate, described for most microorganisms, has recently been proposed. The functionality of this pathway requires the existence of enzymes with prephenate dehydratase (PDT) activity (EC 4.2.1.51) in plants. Using phylogenetic studies, functional complementation assays in yeast and biochemical analysis, we have identified the enzymes displaying PDT activity in Pinus pinaster. Through sequence alignment comparisons and site-directed mutagenesis we have identified a 22-amino acid region conferring PDT activity (PAC domain) and a single Ala314 residue critical to trigger this activity. Our results demonstrate that all plant clades include PAC domain-containing ADTs, suggesting that the PDT activity, and thus the ability to synthesize Phe using phenylpyruvate as an intermediate, has been preserved throughout the evolution of plants. Moreover, this pathway together with the arogenate pathway gives plants a broad and versatile capacity to synthesize Phe and its derived compounds. PAC domain-containing enzymes are also present in green and red algae, and glaucophytes, the three emerging clades following the primary endosymbiont event resulting in the acquisition of plastids in eukaryotes. The evolutionary prokaryotic origin of this domain is discussed. PMID:27125254

  10. Brain activation to briefly presented emotional words: effects of stimulus awareness.

    PubMed

    Hoffmann, Marius; Mothes-Lasch, Martin; Miltner, Wolfgang H R; Straube, Thomas

    2015-02-01

    It is unknown to what extent briefly presented emotional words can be processed without awareness. By means of two independent functional magnetic resonance imaging studies, using either a block or an event-related design, we investigated brain activation to very briefly presented threat related and neutral words during two backward masking conditions (with and without gap between target and mask). In both experiments, emotional words were perceived during the supraliminal "with gap" condition, but they were not recognized during the subliminal "without gap" condition, as indicated by signal detection theory analysis. Imaging results of both experiments showed increased activation of the amygdala, the medial prefrontal cortex and language-processing cortical areas to negative versus neutral words during supraliminal but not subliminal conditions. These results suggest that even very briefly presented emotional words are capable of triggering increased cortical and subcortical processing; however, only when awareness of these stimuli is given. PMID:25324170

  11. Behavior Change Techniques Present in Wearable Activity Trackers: A Critical Analysis

    PubMed Central

    Mercer, Kathryn; Li, Melissa; Giangregorio, Lora; Burns, Catherine

    2016-01-01

    Background Wearable activity trackers are promising as interventions that offer guidance and support for increasing physical activity and health-focused tracking. Most adults do not meet their recommended daily activity guidelines, and wearable fitness trackers are increasingly cited as having great potential to improve the physical activity levels of adults. Objective The objective of this study was to use the Coventry, Aberdeen, and London-Refined (CALO-RE) taxonomy to examine if the design of wearable activity trackers incorporates behavior change techniques (BCTs). A secondary objective was to critically analyze whether the BCTs present relate to known drivers of behavior change, such as self-efficacy, with the intention of extending applicability to older adults in addition to the overall population. Methods Wearing each device for a period of 1 week, two independent raters used CALO-RE taxonomy to code the BCTs of the seven wearable activity trackers available in Canada as of March 2014. These included Fitbit Flex, Misfit Shine, Withings Pulse, Jawbone UP24, Spark Activity Tracker by SparkPeople, Nike+ FuelBand SE, and Polar Loop. We calculated interrater reliability using Cohen's kappa. Results The average number of BCTs identified was 16.3/40. Withings Pulse had the highest number of BCTs and Misfit Shine had the lowest. Most techniques centered around self-monitoring and self-regulation, all of which have been associated with improved physical activity in older adults. Techniques related to planning and providing instructions were scarce. Conclusions Overall, wearable activity trackers contain several BCTs that have been shown to increase physical activity in older adults. Although more research and development must be done to fully understand the potential of wearables as health interventions, the current wearable trackers offer significant potential with regard to BCTs relevant to uptake by all populations, including older adults. PMID:27122452

  12. Atmospheric methane oxidizers are present and active in Canadian high Arctic soils.

    PubMed

    Martineau, Christine; Pan, Yao; Bodrossy, Levente; Yergeau, Etienne; Whyte, Lyle G; Greer, Charles W

    2014-08-01

    The melting of permafrost and the associated potential for methane emissions to the atmosphere are major concerns in the context of global warming. However, soils can also represent a significant sink for methane through the activity of methane-oxidizing bacteria (MOB). In this study, we looked at the activity, diversity, and community structure of MOB at two sampling depths within the active layer in three soils from the Canadian high Arctic. These soils had the capacity to oxidize methane at low (15 ppm) and high (1000 ppm) methane concentrations, but rates differed greatly depending on the sampling date, depth, and site. The pmoA gene sequences related to two genotypes of uncultured MOB involved in atmospheric methane oxidation, the 'upland soil cluster gamma' and the 'upland soil cluster alpha', were detected in soils with near neutral and acidic pH, respectively. Other groups of MOB, including Type I methanotrophs and the 'Cluster 1' genotype, were also detected, indicating a broader diversity of MOB than previously reported for Arctic soils. Overall, the results reported here showed that methane oxidation at both low and high methane concentrations occurs in high Arctic soils and revealed that different groups of atmospheric MOB inhabit these soils.

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

    PubMed

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

    2015-05-20

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

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

    PubMed

    Muramatsu, Akiyoshi

    2016-01-01

    The "School Health and Safety Act" was enforced in April 2009 in Japan, and "school environmental health standards" were established by the Minister of Education, Culture, Sports, Science and Technology. In Article 24 of the Enforcement Regulations, the duties of the school pharmacist have been clarified; school pharmacists have charged with promoting health activities in schools and carrying out complete and regular checks based on the "school environmental health standards" in order to protect the health of students and staff. In supported of this, the school pharmacist group of Japan Pharmaceutical Association has created and distributed digital video discs (DVDs) on "check methods of school environmental health standards" as support material. We use the DVD to ensure the basic issues that school pharmacists deal with, such as objectives, criteria, and methods for each item to be checked, advice, and post-measures. We conduct various workshops and classes, and set up Q&A committees so that inquiries from members are answered with the help of such activities. In addition, school pharmacists try to improve the knowledge of the school staff on environmental hygiene during their in-service training. They also conduct "drug abuse prevention classes" at school and seek to improve knowledge and recognition of drugs, including "dangerous drugs". PMID:27252053

  15. The Construction And Instrumentation Of A Pilot Treatment System At The Standard Mine Superfund Site, Crested Butte, CO - (Presentation)

    EPA Science Inventory

    A pilot biochemical reactor (BCR) was designed and constructed to treat mine-influenced water emanating from an adit at a remote site in southern Colorado which receives an average of 400 inches (10.2 meters) of snowfall each season. The objective of the study is to operate and ...

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

    PubMed

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

    2015-07-17

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

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

    PubMed

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

    2015-07-17

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

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

    PubMed Central

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

    2015-01-01

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

  19. 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. PMID:27186960

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

    PubMed

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

    2015-05-29

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

  1. Annealing of Cd-implanted GaAs: Defect removal, lattice site occupation, and electrical activation

    NASA Astrophysics Data System (ADS)

    Moriya, N.; Brener, I.; Kalish, R.; Pfeiffer, W.; Deicher, M.; Keller, R.; Magerle, R.; Recknagel, E.; Skudlik, H.; Wichert, Th.; Wolf, H.

    1993-05-01

    A systematic investigation of the behavior of Cd-implanted GaAs after rapid thermal annealing is presented. The use of various experimental techniques gives a detailed picture regarding the annealing process in the low-dose regime (1012 and 1013 cm-2) on a microscopic as well as on a macroscopic scale. Perturbed angular correlation experiments, using the radioactive probe 111mCd, yield information on the immediate environment of the Cd implant on an atomic scale. Rutherford backscattering channeling and photoluminescence spectroscopy give complementary information concerning the overall damage level in the implanted layer, Hall measurements are used to determine the degree of electrical activation of the implanted Cd acceptors. The outdiffusion of the implanted radioactive Cd atoms is also investigated. The removal of defects in the next-nearest neighborhood of the Cd atoms takes place after annealing at 700 K and is accompanied by a general recovering of the crystal lattice. Between 600 and 900 K more distant defects are removed. The observed outdiffusion of about one-third of the dopant atoms after annealing above 600 K is discussed in context with their partial incorporation in extended defects. Although already at 700 K, 80% of the implanted Cd atoms are on substitutional lattice sites with no defects in their immediate environment, an annealing temperature in excess of 1000 K is necessary to obtain electrical activation of the implants. It is concluded that compensating defects, present in ion-implanted GaAs, are the reason for the significantly higher temperature required for electrical activation as compared to the incorporation of the dopants on defect-free, substitutional lattice sites.

  2. The Self-Presentation Motives for Physical Activity Questionnaire: Instrument Development and Preliminary Construct Validity Evidence.

    PubMed

    Howle, Timothy C; Dimmock, James A; Whipp, Peter R; Jackson, Ben

    2015-06-01

    With the aim of advancing the literature on impression management in physical activity settings, we developed a theoretically derived 2 by 2 instrument that was designed to measure different types of context-specific self-presentation motives. Following item generation and expert review (Study 1), the instrument was completed by 206 group exercise class attendees (Study 2) and 463 high school physical education students (Study 3). Our analyses supported the intended factor structure (i.e., reflecting acquisitive-agentic, acquisitive-communal, protective-agentic, and protective-communal motives). We found some support for construct validity, and the self-presentation motives were associated with variables of theoretical and applied interest (e.g., impression motivation and construction, social anxiety, social and achievement goals, efficacy beliefs, engagement). Taken together, the results indicate that the Self-presentation Motives for Physical Activity Questionnaire (SMPAQ) may be useful for measuring various types of self-presentation motives in physical activity settings. PMID:26265337

  3. Fractions of aqueous and methanolic extracts from tomato (Solanum lycopersicum L.) present platelet antiaggregant activity.

    PubMed

    Fuentes, Eduado J; Astudillo, Luis A; Gutiérrez, Margarita I; Contreras, Samuel O; Bustamante, Luis O; Rubio, Pia I; Moore-Carrasco, Rodrigo; Alarcón, Marcelo A; Fuentes, Jaime A; González, Daniel E; Palomo, Iván F

    2012-03-01

    Cardiovascular disease (CVD) is the leading cause of death worldwide. Its prevention emphasizes three aspects: not smoking, physical activity and a healthy diet. Recently, we screened the antithrombotic activity of a selected group of fruits and vegetables. Among them, tomato showed an important effect. The aim of this study was to evaluate and characterize the platelet antiaggregatory activity of tomato (Solanum lycopersicum L.). For this, we obtained aqueous and methanolic tomato extracts and evaluated the effect of pH (2 and 10) and temperature (22, 60 and 100°C) on this activity. Furthermore, in order to isolate the antiaggregant principle, we separated tomato extracts into several fractions (A-D) by size exclusion chromatography. In addition, we evaluated the platelet antiaggregating activity ex vivo in Wistar rats. Aqueous and methanolic extracts of tomato treated at 22, 60 and 100°C and pH 2 and 10 still inhibited platelet aggregation (in vitro). Moreover, it was noted that one of the fractions (fraction C), from both aqueous and methanolic extracts, presented the highest activity (∼70% inhibition of platelet aggregation) and concentration dependently inhibited platelet aggregation significantly compared with control (P < 0.05). These fractions did not contain lycopene but presented two peaks of absorption, at 210 and 261 nm, compatible with the presence of nucleosides. In rats treated with tomato macerates, a mild platelet antiaggregating effect ex vivo was observed. Further studies are required to identify the molecules with platelet antiaggregating activity and antiplatelet mechanisms of action. PMID:22185934

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

    PubMed

    Matsubu, John C; Yang, Vanessa N; Christopher, Phillip

    2015-03-01

    CO2 reduction by H2 on heterogeneous catalysts is an important class of reactions that has been studied for decades. However, atomic scale details of structure-function relationships are still poorly understood. Particularly, it has been suggested that metal particle size plays a unique role in controlling the stability of CO2 hydrogenation catalysts and the distribution of active sites, which dictates reactivity and selectivity. These studies often have not considered the possible role of isolated metal active sites in the observed dependences. Here, we utilize probe molecule diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) with known site-specific extinction coefficients to quantify the fraction of Rh sites residing as atomically dispersed isolated sites (Rhiso), as well as Rh sites on the surface of Rh nanoparticles (RhNP) for a series of TiO2 supported Rh catalysts. Strong correlations were observed between the catalytic reverse water gas shift turn over frequency (TOF) and the fraction of Rhiso sites and between catalytic methanation TOF and the fraction of RhNP sites. Furthermore, it was observed that reaction condition-induced disintegration of Rh nanoparticles, forming Rhiso active sites, controls the changing reactivity with time on stream. This work demonstrates that isolated atoms and nanoparticles of the same metal on the same support can exhibit uniquely different catalytic selectivity in competing parallel reaction pathways and that disintegration of nanoparticles under reaction conditions can play a significant role in controlling stability.

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

    PubMed

    Matsubu, John C; Yang, Vanessa N; Christopher, Phillip

    2015-03-01

    CO2 reduction by H2 on heterogeneous catalysts is an important class of reactions that has been studied for decades. However, atomic scale details of structure-function relationships are still poorly understood. Particularly, it has been suggested that metal particle size plays a unique role in controlling the stability of CO2 hydrogenation catalysts and the distribution of active sites, which dictates reactivity and selectivity. These studies often have not considered the possible role of isolated metal active sites in the observed dependences. Here, we utilize probe molecule diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) with known site-specific extinction coefficients to quantify the fraction of Rh sites residing as atomically dispersed isolated sites (Rhiso), as well as Rh sites on the surface of Rh nanoparticles (RhNP) for a series of TiO2 supported Rh catalysts. Strong correlations were observed between the catalytic reverse water gas shift turn over frequency (TOF) and the fraction of Rhiso sites and between catalytic methanation TOF and the fraction of RhNP sites. Furthermore, it was observed that reaction condition-induced disintegration of Rh nanoparticles, forming Rhiso active sites, controls the changing reactivity with time on stream. This work demonstrates that isolated atoms and nanoparticles of the same metal on the same support can exhibit uniquely different catalytic selectivity in competing parallel reaction pathways and that disintegration of nanoparticles under reaction conditions can play a significant role in controlling stability. PMID:25671686

  6. Extending the Diffuse Layer Model of Surface Acidity Behavior: III. Estimating Bound Site Activity Coefficients

    EPA Science Inventory

    Although detailed thermodynamic analyses of the 2-pK diffuse layer surface complexation model generally specify bound site activity coefficients for the purpose of accounting for those non-ideal excess free energies contributing to bound site electrochemical potentials, in applic...

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  8. 77 FR 5560 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

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

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

    NASA Astrophysics Data System (ADS)

    Qi, Jian-Xun; Jiang, Fan

    2011-05-01

    The crystallographic temperature factors (B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule. Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents. It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme. To test the theory of conformational flexibility of enzyme active site, crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions. It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M. It was also found that the B factors averaged over individually collected data sets were more accurate. Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets. Furthermore, we found that the correctly predicted active sites included not only the more flexible residues, but also some more rigid residues. Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network, covering the majority of the substrate binding residues. Therefore, this experimental prediction method may be useful for characterizing the binding site and the function of a protein, such as drug targeting.

  10. Hydrogen production by the naked active site of the di-iron hydrogenases in water.

    PubMed

    Zipoli, Federico; Car, Roberto; Cohen, Morrel H; Selloni, Annabella

    2009-10-01

    We explored the reactivity of the active center of the [FeFe]-hydrogenases detached from the enzyme and immersed in acidified water by first-principles Car-Parrinello molecular-dynamics simulations. We focused on the identification of the structures that are stable and metastable in acidified water and on their activity for hydrogen production. Our calculations revealed that the naked active center could be an efficient catalyst provided that electrons are transferred to the cluster. We found that both bridging and terminal isomers are present at equilibrium and that the bridging configuration is essential for efficient hydrogen production. The formation of the hydrogen molecule occurs via sequential protonations of the distal iron and of the N-atom of the S-CH(2)-NH-CH(2)-S chelating group. H(2) desorption does not involve a significant energy barrier, making the process very efficient at room temperature. We established that the bottleneck in the reaction is the direct proton transfer from water to the vacant site of the distal iron. Moreover, we found that even if the terminal isomer is present at the equilibrium, its strong local hydrophobicity prevents poisoning of the cluster. PMID:19737003

  11. Microbial community changes along the active seepage site of one cold seep in the Red Sea

    PubMed Central

    Cao, Huiluo; Zhang, Weipeng; Wang, Yong; Qian, Pei-Yuan

    2015-01-01

    The active seepage of the marine cold seeps could be a critical process for the exchange of energy between the submerged geosphere and the sea floor environment through organic-rich fluids, potentially even affecting surrounding microbial habitats. However, few studies have investigated the associated microbial community changes. In the present study, 16S rRNA genes were pyrosequenced to decipher changes in the microbial communities from the Thuwal seepage point in the Red Sea to nearby marine sediments in the brine pool, normal marine sediments and water, and benthic microbial mats. An unexpected number of reads from unclassified groups were detected in these habitats; however, the ecological functions of these groups remain unresolved. Furthermore, ammonia-oxidizing archaeal community structures were investigated using the ammonia monooxygenase subunit A (amoA) gene. Analysis of amoA showed that planktonic marine habitats, including seeps and marine water, hosted archaeal ammonia oxidizers that differed from those in microbial mats and marine sediments, suggesting modifications of the ammonia oxidizing archaeal (AOA) communities along the environmental gradient from active seepage sites to peripheral areas. Changes in the microbial community structure of AOA in different habitats (water vs. sediment) potentially correlated with changes in salinity and oxygen concentrations. Overall, the present results revealed for the first time unanticipated novel microbial groups and changes in the ammonia-oxidizing archaea in response to environmental gradients near the active seepages of a cold seep. PMID:26284035

  12. Hydrogen production by the naked active site of the di-iron hydrogenases in water.

    PubMed

    Zipoli, Federico; Car, Roberto; Cohen, Morrel H; Selloni, Annabella

    2009-10-01

    We explored the reactivity of the active center of the [FeFe]-hydrogenases detached from the enzyme and immersed in acidified water by first-principles Car-Parrinello molecular-dynamics simulations. We focused on the identification of the structures that are stable and metastable in acidified water and on their activity for hydrogen production. Our calculations revealed that the naked active center could be an efficient catalyst provided that electrons are transferred to the cluster. We found that both bridging and terminal isomers are present at equilibrium and that the bridging configuration is essential for efficient hydrogen production. The formation of the hydrogen molecule occurs via sequential protonations of the distal iron and of the N-atom of the S-CH(2)-NH-CH(2)-S chelating group. H(2) desorption does not involve a significant energy barrier, making the process very efficient at room temperature. We established that the bottleneck in the reaction is the direct proton transfer from water to the vacant site of the distal iron. Moreover, we found that even if the terminal isomer is present at the equilibrium, its strong local hydrophobicity prevents poisoning of the cluster.

  13. A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells.

    PubMed

    Steimle, Alex; Kalbacher, Hubert; Maurer, Andreas; Beifuss, Brigitte; Bender, Annika; Schäfer, Andrea; Müller, Ricarda; Autenrieth, Ingo B; Frick, Julia-Stefanie

    2016-05-01

    Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory. PMID:26899824

  14. Label free detection of optogenetically stimulated cellular activity by low coherence interferometry (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Satpathy, Sarmishtha; Batabyal, Subrata; Dave, Digant P.; Mohanty, Samarendra K.

    2016-03-01

    Detecting cellular activity in sub-millisecond timescale and micrometer resolution without using invasive means has been a long standing goal in the study of cellular networks. Here, we have employed phase sensitive low coherence interferometry for detecting optogenetically stimulated activity of cells. Nanoscale changes in optical path length (due to change in refractive index and changes in cell thickness) occur when cells are activated, which we aim to detect by phase sensitive low coherence interferometry. A low coherence interferometry and patch-clamp electrophysiology systems were integrated with an inverted fluorescence microscope. Blue laser beam was coupled to the electrophysiology-interferometric detection system for optogenetic stimulation. The phase-sensitive measurements were carried out on Channelrhodopsin-2 sensitized cells (identified by YFP fluorescence) as well as control cells in reflection mode for different intensities and exposures of optogenetic stimulation beam. This method offers good temporal and spatial resolution without using exogenous labeling. Results of studies on all optical stimulation and detection of cellular activity will be presented. Interpretation of the optical activity signals will be discussed in context with changes in cell physiology during stimulation. We will also discuss the potential sources of various artifacts in optical/electrical detection of cellular activity during optical stimulation.

  15. Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site.

    PubMed

    Zhu, Wen-Jing; Li, Miao; Wang, Xiao-Yun

    2007-12-01

    Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (DeltaH degrees ) was 12.38kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK. PMID:17765964

  16. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    SciTech Connect

    Not Available

    1994-02-01

    This report is a detailed listing of all of the Hazardous Waste activities occurring at Martin Marietta`s K-25 site. Contained herein are hazardous waste notification forms, waste stream reports, generator fee forms and various TSDR reports.

  17. Estimation of protein function using template-based alignment of enzyme active sites

    PubMed Central

    2014-01-01

    Background The accumulation of protein structural data occurs more rapidly than it can be characterized by traditional laboratory means. This has motivated widespread efforts to predict enzyme function computationally. The most useful/accurate strategies employed to date are based on the detection of motifs in novel structures that correspond to a specific function. Functional residues are critical components of predictively useful motifs. We have implemented a novel method, to complement current approaches, which detects motifs solely on the basis of distance restraints between catalytic residues. Results ProMOL is a plugin for the PyMOL molecular graphics environment that can be used to create active site motifs for enzymes. A library of 181 active site motifs has been created with ProMOL, based on definitions published in the Catalytic Site Atlas (CSA). Searches with ProMOL produce better than 50% useful Enzyme Commission (EC) class suggestions for level 1 searches in EC classes 1, 4 and 5, and produce some useful results for other classes. 261 additional motifs automatically translated from Jonathan Barker’s JESS motif set [Bioinformatics 19:1644–1649, 2003] and a set of NMR motifs is under development. Alignments are evaluated by visual superposition, Levenshtein distance and root-mean-square deviation (RMSD) and are reasonably consistent with related search methods. Conclusion The ProMOL plugin for PyMOL provides ready access to template-based local alignments. Recent improvements to ProMOL, including the expanded motif library, RMSD calculations and output selection formatting, have greatly increased the program’s usability and speed, and have improved the way that the results are presented. PMID:24669788

  18. A Lagrangian analysis of the present-day sources of moisture for major ice-core sites

    NASA Astrophysics Data System (ADS)

    Drumond, Anita; Taboada, Erica; Nieto, Raquel; Gimeno, Luis; Vicente-Serrano, Sergio M.; López-Moreno, Juan Ignacio

    2016-07-01

    A Lagrangian approach was used to identify the moisture sources for 14 ice-core sites located worldwide for the period of 1980-2012. The sites were classified into three domains: Arctic, Central (Andes, Alps, and Kilimanjaro), and Antarctic. The approach was used to compute budgets of evaporation minus precipitation by calculating changes in the specific humidity along 10-day backward trajectories. The results indicate that the oceanic regions around the subtropical high-pressure centres provide most of moisture, and their contribution varies throughout the year following the annual cycles of the centres. For the Arctic Domain, the sources lie in the subtropical North Atlantic and Pacific. The subtropical South Atlantic, Indian, and Pacific oceans provide moisture for the Antarctic Domain. The sources for South America are the Atlantic and South Pacific, for Europe the sources are in the Mediterranean and the North Atlantic, and for Asia the sources are the Indian Ocean and the Arabian Sea.

  19. Tales from the Dark Side: Teacher Professional Development , Support , Activities, Student Research & Presentations

    NASA Astrophysics Data System (ADS)

    Walker, C. E.; Pompea, S. M.

    2013-12-01

    In a partnership last Spring with Arizona Public Service, the National Optical Astronomy Observatory (NOAO) created the 'Dark-Skies Energy Education Program: Energy Awareness for a Sustainable Future'. In this program, experienced science and technology education specialists from NOAO led 2 one-day professional development workshops for thirteen 6th grade teachers on dark skies and energy education. The workshops focused on three foundational, scaffolding activities and a final student research project. This in turn culminated in a Family Science Night where students presented their projects. In between these events, our NOAO team provided support for teachers through real-time video conferencing using FaceTime. In addition to the professional development, each teacher received a kit full of resource materials to perform the activities and research project. The kit was at no cost to the teacher, school, or district. Each kit contained the latest version of a tablet, which was used to facilitate communication and support for the teachers, as well as provide all the program's written teaching materials. The activities are in accordance with state, Common Core and Next Generation Science Standards. Our NOAO instructors gave firsthand experiences on how best to use these materials in a classroom or public setting. They also discussed opportunities on how they can incorporate, adapt and expand upon the activities and research projects in the classroom. Evaluation reports from the program's independent evaluator showed that the students enjoyed learning from the three foundational activities and research projects. The project presentations by the Yuma students were outstanding in their creativity, level of effort, and scientific accuracy. To summarize the evaluations, significant changes in knowledge and attitude were made with the teachers and students (from one-on-one interviews and surveys), but behavioral changes (albeit only over a semester) seemed minimal. The AGU

  20. Antigen presentation by non-immune B-cell hybridoma clones: presentation of synthetic antigenic sites reveals clones that exhibit no specificity and clones that present only one epitope

    NASA Technical Reports Server (NTRS)

    Cohly, H. H.; Morrison, D. R.; Atassi, M. Z.

    1989-01-01

    Recently, we reported the preparation and antigen-presenting properties of hybridoma B-cell clones obtained after fusing non-secreting, non-antigen presenting Balb/c 653-myeloma cells with non-immune SJL spleen cells. It was found that antigen presentation at the clonal level can be specific or non-specific, depending on the particular B-cell clone. In the present work, one specific and one general presenter B-cell clones were tested for their epitope presentation ability to SJL T-cells that were specific to lysozyme or myoglobin. B-cell clone A1G12, a general presenter which presented both lysozyme and myoglobin to their respective T-cell lines, was found to present all five myoglobin epitopes while clone A1L16, a lysozyme specific presenter presented only one of the three epitopes of lysozyme. The latter reveals a hitherto unknown submolecular specificity (to a given epitope within a protein) for antigen presenting cells at the clonal level. Therefore, the specificity of T-cell recognition does not only derive from the T-cell but may also be dependent on the epitope specificity of the antigen-presenting B-cell.

  1. Quantum dot based enzyme activity sensors present deviations from Michaelis-Menten kinetic model

    NASA Astrophysics Data System (ADS)

    Díaz, Sebastián. A.; Brown, Carl W.; Malanoski, Anthony P.; Oh, Eunkeu; Susumu, Kimihiro; Medintz, Igor L.

    2016-03-01

    Nanosensors employing quantum dots (QDs) and enzyme substrates with fluorescent moieties offer tremendous promise for disease surveillance/diagnostics and as high-throughput co-factor assays. Advantages of QDs over other nanoscaffolds include their small size and inherent photochemical properties such as size tunable fluorescence, ease in attaching functional moieties, and resistance to photobleaching. These properties make QDs excellent Förster Resonance Energy Transfer (FRET) donors; well-suited for rapid, optical measurement applications. We report enzyme sensors designed with a single FRET donor, the QD donor acting as a scaffold to multiple substrates or acceptors. The QD-sensor follows the concrete activity of the enzyme, as compared to the most common methodologies that quantify the enzyme amount or its mRNA precursor. As the sensor reports on the enzyme activity in real-time we can actively follow the kinetics of the enzyme. Though classic Michaelis-Menten (MM) parameters can be obtained to describe the activity. In the course of these experiments deviations, both decreasing and increasing the kinetics, from the common MM model were observed upon close examinations. From these observations additional experiments were undertaken to understand the varying mechanisms. Different enzymes can present different deviations depending on the chosen target, e.g. trypsin appears to present a positive hopping mechanism while collagenase demonstrates a QD caused reversible inhibition.

  2. Anisotropic Covalency Contributions to Superexchange Pathways in Type One Copper Active Sites

    PubMed Central

    2015-01-01

    Type one (T1) Cu sites deliver electrons to catalytic Cu active sites: the mononuclear type two (T2) Cu site in nitrite reductases (NiRs) and the trinuclear Cu cluster in the multicopper oxidases (MCOs). The T1 Cu and the remote catalytic sites are connected via a Cys-His intramolecular electron-transfer (ET) bridge, which contains two potential ET pathways: P1 through the protein backbone and P2 through the H-bond between the Cys and the His. The high covalency of the T1 Cu–S(Cys) bond is shown here to activate the T1 Cu site for hole superexchange via occupied valence orbitals of the bridge. This covalency-activated electronic coupling (HDA) facilitates long-range ET through both pathways. These pathways can be selectively activated depending on the geometric and electronic structure of the T1 Cu site and thus the anisotropic covalency of the T1 Cu–S(Cys) bond. In NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being more efficient. Comparing the MCOs to NiRs, the second-sphere environment changes the conformation of the Cys-His pathway, which selectively activates HDA for superexchange by blue π sites for efficient turnover in catalysis. These studies show that a given protein bridge, here Cys-His, provides different superexchange pathways and electronic couplings depending on the anisotropic covalencies of the donor and acceptor metal sites. PMID:25310460

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

    PubMed

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

    2013-12-01

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

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

    PubMed

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

    2014-04-01

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

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

    SciTech Connect

    1987-12-31

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

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

    NASA Astrophysics Data System (ADS)

    Miyamoto, Daisuke; Ichikuni, Nobuyuki; Shimazu, Shogo

    2007-02-01

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

  7. Screening breeding sites of the common toad (Bufo bufo) in England and Wales for evidence of endocrine disrupting activity.

    PubMed

    Pickford, Daniel B; Jones, Alexandra; Velez-Pelez, Alejandra; Orton, Frances; Iguchi, Taisen; Mitsui, Naoko; Tooi, Osamu

    2015-07-01

    Anuran amphibians are often present in agricultural landscapes and may therefore be exposed to chemicals in surface waters used for breeding. We used passive accumulation devices (SPMD and POCIS) to sample contaminants from nine breeding sites of the Common toad (Bufo bufo) across England and Wales, measuring endocrine activity of the extracts in a recombinant yeast androgen screen (YAS) and yeast estrogen screen (YES) and an in vitro vitellogenin induction screen in primary culture of Xenopus laevis hepatocytes. We also assessed hatching, growth, survival, and development in caged larvae in situ, and sampled metamorphs for gonadal histopathology. None of the SPMD extracts exhibited estrogen receptor or androgen receptor agonist activity, while POCIS extracts from two sites in west-central England exhibited concentration-dependent androgenic activity in the YAS. Three sites exhibited significant estrogenic activity in both the YES and the Xenopus hepatocyte. Hatching rates varied widely among sites, but there was no consistent correlation between hatching rate and intensity of agricultural activity, predicted concentrations of agrochemicals, or endocrine activity measured in YES/YAS assays. While a small number of intersex individuals were observed, their incidence could not be associated with predicted pesticide exposure or endocrine activitity measured in the in vitro screens. There were no significant differences in sex ratio, as determined by gonadal histomorphology among the study sites, and no significant correlation was observed between proportion of males and predicted exposure to agrochemicals. However, a negative correlation did become apparent in later sampling periods between proportion of males and estrogenic activity of the POCIS sample, as measured in the YES. Our results suggest that larval and adult amphibians may be exposed to endocrine disrupting chemicals in breeding ponds, albeit at low concentrations, and that chemical contaminants other than

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

    SciTech Connect

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

    1991-11-01

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

  9. Active sites without restraints: high-resolution analysis of metal cofactors.

    PubMed

    Burger, Eva-Maria; Andrade, Susana L A; Einsle, Oliver

    2015-12-01

    For most three-dimensional structures of biological macromolecules, the factual accuracy of atom positions by far exceeds the resolution of the experimental data, although the refinement problem presented by a protein structure is substantially underdetermined. This is achieved through using restraints that precisely define protein geometries and thus reduce the degrees of freedom of the refinement problem. If such information is not available or when unusual geometries or particular ligand states complicate structural analysis, possible pitfalls arise that not only concern the precise definition of spatial arrangements, but also the identification of atom types and bond distances. Prominent examples include CO dehydrogenase, hydrogenase, acetylene hydratase and nitrogenase, all of which employ unique active sites that turned out not to be what they seemed upon first inspection.

  10. Regulatory impact analysis of environmental standards for uranium mill tailings at active sites. Final report

    SciTech Connect

    Not Available

    1983-03-01

    The Environmental Protection Agency was directed by Congress, under PL 95-604, the Uranium Mill Tailings Radiation Control Act of 1978, to set standards of general application that provide protection from the hazards associated with uranium mill tailings. Title I of the Act pertains to tailings at inactive sites for which the Agency has developed standards as part of a separate rulemaking. Title II of the Act requires standards covering the processing and disposal of byproduct materials at mills which are currently licensed by the appropriate regulatory authorities. This Regulatory Impact Analysis (RIA) addresses the standards developed under Title II. There are two major parts of the standards for active mills: standards for control of releases from tailings during processing operations and prior to final disposal, and standards for protection of the public after the disposal of tailings. This report presents a detailed analysis of standards for disposal only, since the analysis required for the operations standards is very limited.

  11. Implication of crystal water molecules in inhibitor binding at ALR2 active site.

    PubMed

    Hymavati; Kumar, Vivek; Sobhia, M Elizabeth

    2012-01-01

    Water molecules play a crucial role in mediating the interaction between a ligand and a macromolecule. The solvent environment around such biomolecule controls their structure and plays important role in protein-ligand interactions. An understanding of the nature and role of these water molecules in the active site of a protein could greatly increase the efficiency of rational drug design approaches. We have performed the comparative crystal structure analysis of aldose reductase to understand the role of crystal water in protein-ligand interaction. Molecular dynamics simulation has shown the versatile nature of water molecules in bridge H bonding during interaction. Occupancy and life time of water molecules depend on the type of cocrystallized ligand present in the structure. The information may be useful in rational approach to customize the ligand, and thereby longer occupancy and life time for bridge H-bonding. PMID:22649481

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

    PubMed

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

    2012-02-01

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

  13. Regulation of protein synthesis and autophagy in activated dendritic cells: implications for antigen processing and presentation.

    PubMed

    Argüello, Rafael J; Reverendo, Marisa; Gatti, Evelina; Pierre, Philippe

    2016-07-01

    Antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules originate from the degradation of both self and non-self proteins. T cells can therefore recognize at the surface of surveyed cells, the self-peptidome produced by the cell itself (mostly inducing tolerance) or immunogenic peptides derived from exogenous origins. The initiation of adaptive immune responses by dendritic cells (DCs), through the antigenic priming of naïve T cells, is associated to microbial pattern recognition receptors engagement. Activation of DCs by microbial product or inflammatory cytokines initiates multiple processes that maximize DC capacity to present exogenous antigens and stimulate T cells by affecting major metabolic and membrane traffic pathways. These include the modulation of protein synthesis, the regulation of MHC and co-stimulatory molecules transport, as well as the regulation of autophagy, that, all together promote exogenous antigen presentation while limiting the display of self-antigens by MHC molecules.

  14. Coordination number of zinc ions in the phosphotriesterase active site by molecular dynamics and quantum mechanics.

    PubMed

    Koca, Jaroslav; Zhan, Chang-Guo; Rittenhouse, Robert C; Ornstein, Rick L

    2003-02-01

    We have run several molecular dynamics (MD) simulations on zinc-containing phosphotriesterase (PTE) with two bound substrates, sarin and paraoxon, and with the substrate analog diethyl 4-methylbenzylphosphonate. A standard nonbonded model was employed to treat the zinc ions with the commonly used charge of +2. In all the trajectories, we observed a tightly bound water (TBW) molecule in the active site that was coordinated to the less buried zinc ion. The phosphoryl oxygen of the substrate/inhibitor was found to be coordinated to the same zinc ion so that, considering all ligands, the less buried zinc was hexa-coordinated. The hexa-coordination of this zinc ion was not seen in the deposited X-ray pdb files for PTE. Several additional MD simulations were then performed using different charges (+1, +1.5) on the zinc ions, along with ab initio and density functional theory (DFT) calculations, to evaluate the following possibilities: the crystal diffraction data were not correctly interpreted; the hexa-coordinated zinc ion in PTE is only present in solution and not in the crystal; and the hexa-coordinated zinc ion in PTE is an artifact of the force field used. A charge of +1.5 leads to a coordination number (CN) of 5 on both zinc ions, which is consistent with the results from ab initio and DFT calculations and with the latest high resolution X-ray crystal structure. The commonly used charge of +2 produces a CN of 6 on the less buried zinc. The CN on the more buried zinc ion is 5 when the substrate/inhibitor is present in the simulation, and increases to 6 when the substrate/inhibitor is removed prior to the simulation. The results of both of the MD and quantum mechanical calculations lead to the conclusion that the zinc ions in the PTE active site are both penta-coordinated, and that the MD simulations performed with the charge of +2 overestimate the CN of the zinc ions in the PTE active site. The overall protein structures in the simulations remain unaffected by the

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

    PubMed

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

    2016-09-01

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

  16. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    SciTech Connect

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B.; Sagi, Irit; Havenith, Martina

    2011-09-18

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

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

    PubMed

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

    2010-09-01

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

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

    SciTech Connect

    Held, Jeanette Smaalen, Sander van

    2014-04-01

    Chemical bonding at the active site of lysozyme is analyzed on the basis of a multipole model employing transferable multipole parameters from a database. Large B factors at low temperatures reflect frozen-in disorder, but therefore prevent a meaningful free refinement of multipole parameters. Chemical bonding at the active site of hen egg-white lysozyme (HEWL) is analyzed on the basis of Bader’s quantum theory of atoms in molecules [QTAIM; Bader (1994 ▶), Atoms in Molecules: A Quantum Theory. Oxford University Press] applied to electron-density maps derived from a multipole model. The observation is made that the atomic displacement parameters (ADPs) of HEWL at a temperature of 100 K are larger than ADPs in crystals of small biological molecules at 298 K. This feature shows that the ADPs in the cold crystals of HEWL reflect frozen-in disorder rather than thermal vibrations of the atoms. Directly generalizing the results of multipole studies on small-molecule crystals, the important consequence for electron-density analysis of protein crystals is that multipole parameters cannot be independently varied in a meaningful way in structure refinements. Instead, a multipole model for HEWL has been developed by refinement of atomic coordinates and ADPs against the X-ray diffraction data of Wang and coworkers [Wang et al. (2007), Acta Cryst. D63, 1254–1268], while multipole parameters were fixed to the values for transferable multipole parameters from the ELMAM2 database [Domagala et al. (2012), Acta Cryst. A68, 337–351] . Static and dynamic electron densities based on this multipole model are presented. Analysis of their topological properties according to the QTAIM shows that the covalent bonds possess similar properties to the covalent bonds of small molecules. Hydrogen bonds of intermediate strength are identified for the Glu35 and Asp52 residues, which are considered to be essential parts of the active site of HEWL. Furthermore, a series of weak C

  19. Dried extracts of Encholirium spectabile (Bromeliaceae) present antioxidant and photoprotective activities in vitro

    PubMed Central

    de Oliveira, Raimundo Gonçalves; Souza, Grasielly Rocha; Guimarães, Amanda Leite; de Oliveira, Ana Paula; Silva Morais, Amanda Caroline; da Cruz Araújo, Edigênia Cavalcante; Nunes, Xirley Pereira; Almeida, Jackson Roberto Guedes da Silva

    2013-01-01

    The antioxidant and photoprotective activities of dried extracts from the leaves of Encholirium spectabile were investigated. It was also evaluated the total phenolic and flavonoid contents by the Folin–Ciocalteu and aluminum chloride methods, respectively. Antioxidant activities of the extracts were evaluated by using of 2,2-diphenyl-1-picrylhydrazil (DPPH) radical scavenging and β-carotene–linoleic acid bleaching and compared with ascorbic acid, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) used as reference compounds. The photoprotective effect was evaluated by the spectrophotometric method. The most significant total phenolic and flavonoid contents was of 188.50 ± 27.50 mg of gallic acid equivalent/g and 129.70 ± 4.59 mg of catechin equivalent/g, respectively, for chloroform fraction (Es-CHCl3). The Es-CHCl3 also presented the best antioxidant activity (IC50 25.35 ± 4.35 μg/ml) for DPPH scavenging. The ethanol extract (Es-EtOH), Es-CHCl3 and the fraction ethyl acetate (Es-AcOEt) showed characteristic absorption bands in regions UVB and UVA in a concentration-dependent manner. Es-CHCl3 presented the highest sun protection factor SPF (8.89 ± 2.11). It shows the possibility to use this extract as sunscreen in pharmaceutical preparations. PMID:24396251

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

    SciTech Connect

    Crichlow, G.; Lubetsky, J; Leng, L; Bucala, R; Lolis, E

    2009-01-01

    Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

  1. The modality-switch effect: visually and aurally presented prime sentences activate our senses.

    PubMed

    Scerrati, Elisa; Baroni, Giulia; Borghi, Anna M; Galatolo, Renata; Lugli, Luisa; Nicoletti, Roberto

    2015-01-01

    Verifying different sensory modality properties for concepts results in a processing cost known as the modality-switch effect. It has been argued that this cognitive cost is the result of a perceptual simulation. This paper extends this argument and reports an experiment investigating whether the effect is the result of an activation of sensory information which can also be triggered by perceptual linguistically described stimuli. Participants were first exposed to a prime sentence describing a light or a sound's perceptual property (e.g., "The light is flickering", "The sound is echoing"), then required to perform a property-verification task on a target sentence (e.g., "Butter is yellowish", "Leaves rustle"). The content modalities of the prime and target sentences could be compatible (i.e., in the same modality: e.g., visual-visual) or not (i.e., in different modalities). Crucially, we manipulated the stimuli's presentation modality such that half of the participants was faced with written sentences while the other half was faced with aurally presented sentences. Results show a cost when two different modalities alternate, compared to when the same modality is repeated with both visual and aural stimuli presentations. This result supports the embodied and grounded cognition view which claims that conceptual knowledge is grounded into the perceptual system. Specifically, this evidence suggests that sensory modalities can be pre-activated through the simulation of either read or listened linguistic stimuli describing visual or acoustic perceptual properties.

  2. AKAP9 regulates activation-induced retention of T lymphocytes at sites of inflammation

    PubMed Central

    Herter, Jan M.; Grabie, Nir; Cullere, Xavier; Azcutia, Veronica; Rosetti, Florencia; Bennett, Paul; Herter-Sprie, Grit S.; Elyaman, Wassim; Luscinskas, Francis W.; Lichtman, Andrew H.; Mayadas, Tanya N.

    2015-01-01

    The mechanisms driving T cell homing to lymph nodes and migration to tissue are well described but little is known about factors that affect T cell egress from tissues. Here, we generate mice with a T cell-specific deletion of the scaffold protein A kinase anchoring protein 9 (AKAP9) and use models of inflammatory disease to demonstrate that AKAP9 is dispensable for T cell priming and migration into tissues and lymph nodes, but is required for T cell retention in tissues. AKAP9 deficiency results in increased T cell egress to draining lymph nodes, which is associated with impaired T cell re-activation in tissues and protection from organ damage. AKAP9-deficient T cells exhibit reduced microtubule-dependent recycling of TCRs back to the cell surface and this affects antigen-dependent activation, primarily by non-classical antigen-presenting cells. Thus, AKAP9-dependent TCR trafficking drives efficient T cell re-activation and extends their retention at sites of inflammation with implications for disease pathogenesis. PMID:26680259

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

    PubMed

    Kudernatsch, Wilhelmine; Peng, Guowen; Zeuthen, Helene; Bai, Yunhai; Merte, Lindsay R; Lammich, Lutz; Besenbacher, Flemming; Mavrikakis, Manos; Wendt, Stefan

    2015-08-25

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

  4. Analysis of Chlamydia caviae entry sites and involvement of Cdc42 and Rac activity.

    PubMed

    Subtil, Agathe; Wyplosz, Benjamin; Balañá, María Eugenia; Dautry-Varsat, Alice

    2004-08-01

    In epithelial cells, endocytic activity is mostly dedicated to nutrient and macromolecule uptake. To invade these cells, Chlamydiaceae, like other pathogens, have evolved strategies that utilise the existing endocytic machineries and signalling pathways, but little is known about the host cell molecules involved. In this report, we show that within five minutes of infection of HeLa cells by Chlamydia caviae GPIC strain several events take place in the immediate vicinity of invasive bacteria: GM1-containing microdomains cluster, tyrosine-phosphorylated proteins accumulate, and intense actin polymerization occurs. We show that actin polymerization is controlled by the small GTPases Cdc42 and Rac, which become activated upon infection. Expression of dominant negative forms of these GTPases inhibits C. caviae entry and leads to abnormal actin polymerization. In contrast, the small GTPase Rho does not seem essential for bacterial entry. Finally, phosphatidylinositol 3-kinase activity is also required for internalization of C. caviae, probably downstream of the other molecular events reported here. We present the first scheme of the events occurring at the sites of invasion of epithelial cells by a member of the Chlamydiaceae family.

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

    SciTech Connect

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

    2015-08-25

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

  6. Nanoscale electrochemical patterning reveals the active sites for catechol oxidation at graphite surfaces.

    PubMed

    Patel, Anisha N; McKelvey, Kim; Unwin, Patrick R

    2012-12-19

    Graphite-based electrodes (graphite, graphene, and nanotubes) are used widely in electrochemistry, and there is a long-standing view that graphite step edges are needed to catalyze many reactions, with the basal surface considered to be inert. In the present work, this model was tested directly for the first time using scanning electrochemical cell microscopy reactive patterning and shown to be incorrect. For the electro-oxidation of dopamine as a model process, the reaction rate was measured at high spatial resolution across a surface of highly oriented pyrolytic graphite. Oxidation products left behind in a pattern defined by the scanned electrochemical cell served as surface-site markers, allowing the electrochemical activity to be correlated directly with the graphite structure on the nanoscale. This process produced tens of thousands of electrochemical measurements at different locations across the basal surface, unambiguously revealing it to be highly electrochemically active, with step edges providing no enhanced activity. This new model of graphite electrodes has significant implications for the design of carbon-based biosensors, and the results are additionally important for understanding electrochemical processes on related sp(2)-hybridized materials such as pristine graphene and nanotubes.

  7. AKAP9 regulates activation-induced retention of T lymphocytes at sites of inflammation.

    PubMed

    Herter, Jan M; Grabie, Nir; Cullere, Xavier; Azcutia, Veronica; Rosetti, Florencia; Bennett, Paul; Herter-Sprie, Grit S; Elyaman, Wassim; Luscinskas, Francis W; Lichtman, Andrew H; Mayadas, Tanya N

    2015-01-01

    The mechanisms driving T cell homing to lymph nodes and migration to tissue are well described but little is known about factors that affect T cell egress from tissues. Here, we generate mice with a T cell-specific deletion of the scaffold protein A kinase anchoring protein 9 (AKAP9) and use models of inflammatory disease to demonstrate that AKAP9 is dispensable for T cell priming and migration into tissues and lymph nodes, but is required for T cell retention in tissues. AKAP9 deficiency results in increased T cell egress to draining lymph nodes, which is associated with impaired T cell re-activation in tissues and protection from organ damage. AKAP9-deficient T cells exhibit reduced microtubule-dependent recycling of TCRs back to the cell surface and this affects antigen-dependent activation, primarily by non-classical antigen-presenting cells. Thus, AKAP9-dependent TCR trafficking drives efficient T cell re-activation and extends their retention at sites of inflammation with implications for disease pathogenesis. PMID:26680259

  8. Present-day biogeochemical activities of anaerobic bacteria and their relevance to future exobiological investigations

    USGS Publications Warehouse

    Oremland, R.S.

    1989-01-01

    If the primordial atmosphere was reducing, then the first microbial ecosystem was probably composed of anaerobic bacteria. However, despite the presence of an oxygen-rich atmosphere, anaerobic habitats are important, commonplace components of the Earth's present biosphere. The geochemical activities displayed by these anaerobes impact the global cycling of certain elements (e.g., C, N, S, Fe, Mn, etc.). Methane provides an obvious example of how human-enhanced activities on a global scale can influence the content of a "radiative" (i.e., infrared absorbing) trace gas in the atmosphere. Methane can be oxidized by anaerobic bacteria, but this does not appear to support their growth. Acetylene, however, does support such growth. This may form the basis for future exobiological investigations of the atmospheres of anoxic, hydrocarbon-rich planets like Jupiter and Saturn, as well as the latter's satellite Titan. ?? 1989.

  9. [Plasmapheresis for macrophage activation syndrome and multiorgan failure as first presentation of juvenile dermatomyositis].

    PubMed

    Bustos B, R; Carrasco A, C; Toledo R, C

    2012-07-01

    The use of extracorporeal techniques for the treatment of paediatric diseases has expanded dramatically in the past decade. Plasmapheresis, a technique for exchanging plasma components with albumin or plasma, has been used in some rheumatologic conditions. We report the clinical course of a 7 years old boy with clinical and biological features of macrophage activation syndrome and multiorgan failure, at the time of presentation of severe juvenile dermatomyositis, and non responsive to corticosteroids, cyclosporine and immunoglobulin. After 4 days in the paediatric intensive care unit, plasmapheresis was used as rescue therapy. Repeated therapeutic plasmapheresis was effective for improving the multiorgan failure and laboratory abnormalities. The patient was discharged on the 21st hospital day with good functional condition. Plasmapheresis should be considered as rescue treatment in patients with life threatening macrophage activation syndrome and systemic onset of juvenile dermatomyositis. PMID:22342516

  10. Peptides from cowpea present antioxidant activity, inhibit cholesterol synthesis and its solubilisation into micelles.

    PubMed

    Marques, Marcelo Rodrigues; Soares Freitas, Rosana Aparecida Manólio; Corrêa Carlos, Amanda Caroline; Siguemoto, Érica Sayuri; Fontanari, Gustavo Guadagnucci; Arêas, José Alfredo Gomes

    2015-02-01

    In previous studies, it was reported that the protein isolated from the cowpea interferes favourably in lipid metabolism, and reduces cholesterol synthesis. The present study investigated the role of cowpea peptide fractions in the micellar solubilisation of cholesterol, in the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) activity, and in the in vitro antioxidant capacity, considering the effects of thermal processing. The protein was isolated from the raw and cooked beans and digested to simulate human digestion. The peptides from the protein isolate of raw bean with molecular mass lower than 3kDa reduced 89% of the HMGCR enzymatic reaction velocity. The cooked cowpeas were more effective in inhibiting the micellar solubility of cholesterol than the raw ones but not the antioxidant activity. This is the first report that cowpea peptides inhibit cholesterol homeostasis in vitro in two distinct routes, and act as an antioxidant.

  11. Differential diagnosis of double site intussusception in childhood: a 15-year-old girl presenting with bowel obstruction.

    PubMed

    Davidson, Joseph; Wright, Naomi Jane; Kufeji, Dorothy

    2015-01-01

    Abdominal pain in a teenage girl is common; however, thorough history and examination can often distinguish a more rare or sinister diagnosis. We present a case of a 15-year-old girl presenting with abdominal pain, who was subsequently diagnosed with double intussusception secondary to her previously unrecognised Peutz-Jeghers syndrome (PJS). PMID:26581705

  12. Deciphering the Long-Term Trend of Atlantic Basin Intense Hurricanes: More Active Versus Less Active During the Present Epoch

    NASA Technical Reports Server (NTRS)

    Wilson, Robert M.

    1998-01-01

    During the interval of 1944-1997, 120 intense hurricanes (i.e., those of category 3 or higher on the Saffir-Simpson hurricane damage potential scale) were observed in the Atlantic basin, having an annual frequency of 0-7 events per year, being more active prior to the mid 1960's than thereafter (hence a possible two-state division: more active versus less active), and being preferentially lower during El Nino years as compared to non-El Nino years. Because decadal averages of the frequency of intense hurricanes closely resemble those of average temperature anomalies for northern hemispheric and global standards and of the average temperature at the Armagh Observatory (Northern Ireland), a proxy for climatic change, it is inferred that the long-term trends of the annual frequency of intense hurricanes and temperature may be statistically related. Indeed, on the basis of 4- and 10-yr moving averages, one finds that there exists strong linear associations between the annual frequency of intense hurricanes in the Atlantic basin and temperature (specially, when temperature slightly leads). Because the long-term leading trends of temperature are now decidedly upward, beginning about the mid 1980's, it is inferred that the long-term consequential trends of the annual frequency of intense hurricanes should now also be upward, having begun near 1990, suggesting that a return to the more active state probably has already occurred. However, because of the anomalous El Nino activity of the early to mid 1990's, the switch from the less active to the more active state essentially went unnoticed (a marked increase in the number of intense hurricanes was not observed until the 1995 and 1996 hurricane seasons, following the end of the anomalous El Nino activity). Presuming that a return to the more active state has, indeed, occurred, one expects the number of seasonal intense hurricanes during the present epoch (continuing through about 2012) to usually be higher than average (i

  13. Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site*

    PubMed Central

    Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

    2014-01-01

    Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser105 residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T5015, the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability. PMID:24448805

  14. Video and film analysis with correlation tracking and active result presentation (Abstract Only)

    NASA Astrophysics Data System (ADS)

    Rowa, Per

    1990-08-01

    Experience with a turnkey analysis system featuring high resolution video input and display, a modular video disc system and a 16 mm cine film scanner with 2600-point resolution, is presented. Tracking is performed with a high-speed correlation process, requiring no special markers. Software packages for evaluating two and three-dimensional results are interactively accessible. Combining the original image sequence with real-time graphic overlays and active drawing of graphic diagrams, provides for an excellent understanding and documentation of the motion sequences.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  17. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

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

  18. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  19. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

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

  20. 77 FR 39508 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-03

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

  1. Computational approaches to the determination of active site structures and reaction mechanisms in heterogeneous catalysts.

    PubMed

    Catlow, C R A; French, S A; Sokol, A A; Thomas, J M

    2005-04-15

    We apply quantum chemical methods to the study of active site structures and reaction mechanisms in mesoporous silica and metal oxide catalysts. Our approach is based on the use of both molecular cluster and embedded cluster (QM/MM) techniques, where the active site and molecular complex are described using density functional theory (DFT) and the embedding matrix simulated by shell model potentials. We consider three case studies: alkene epoxidation over the microporous TS-1 catalyst; methanol synthesis on ZnO and Cu/ZnO and C-H bond activation over Li-doped MgO.

  2. Computational approaches to the determination of active site structures and reaction mechanisms in heterogeneous catalysts.

    PubMed

    Catlow, C R A; French, S A; Sokol, A A; Thomas, J M

    2005-04-15

    We apply quantum chemical methods to the study of active site structures and reaction mechanisms in mesoporous silica and metal oxide catalysts. Our approach is based on the use of both molecular cluster and embedded cluster (QM/MM) techniques, where the active site and molecular complex are described using density functional theory (DFT) and the embedding matrix simulated by shell model potentials. We consider three case studies: alkene epoxidation over the microporous TS-1 catalyst; methanol synthesis on ZnO and Cu/ZnO and C-H bond activation over Li-doped MgO. PMID:15901543

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

    PubMed

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

    1998-12-01

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

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

    SciTech Connect

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

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

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

    PubMed Central

    Ping, Z A; Butterfiel, D A

    1991-01-01

    A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and fluorescence results were in agreement and both paralleled enzymatic activity of papain with respect to both the variation in pH and denaturation. These results strongly suggest that SL-PMB and Acrylodan labels can be used to characterize the physical state of the active site of the enzyme. PMID:1657229

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

    PubMed Central

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

    2015-01-01

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis. DOI: http://dx.doi.org/10.7554/eLife.11620.001 PMID:26673079

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

    PubMed

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

    2015-12-16

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.

  8. Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

    PubMed Central

    Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I.; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity. PMID:24278284

  9. HTO and OBT activity concentrations in soil at the historical atmospheric HT release site (Chalk River Laboratories).

    PubMed

    Kim, S B; Bredlaw, M; Korolevych, V Y

    2012-01-01

    Tritium is routinely released by the Chalk River Laboratories (CRL) nuclear facilities. Three International HT release experiments have been conducted at the CRL site in the past. The site has not been disturbed since the last historical atmospheric testing in 1994 and presents an opportunity to assess the retention of tritium in soil. This study is devoted to the measurement of HTO and OBT activity concentration profiles in the subsurface 25 cm of soil. In terms of soil HTO, there is no evidence from the past HT release experiments that HTO was retained. The HTO activity concentration in the soil pore water appears similar to concentrations found in background areas in Ontario. In contrast, OBT activity concentrations in soil at the same site were significantly higher than HTO activity concentrations in soil. Elevated OBT appears to reside in the top layer of the soil (0-5 cm). In addition, OBT activity concentrations in the top soil layer did not fluctuate much with season, again, quite in contrast with soil HTO. This result suggests that OBT activity concentrations retained the signature of the historical tritium releases.

  10. A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

    SciTech Connect

    Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla; Peterson, Scott N.; Caffrey, Michael; Anderson, Wayne F.; Lavie, Arnon

    2012-04-18

    Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.

  11. Structure of HIV-1 Reverse Transcriptase with the Inhibitor -thujaplicinol Bound at the RNase H Active Site

    SciTech Connect

    Himmel, D.; Maegley, K; Pauly, T; Bauman, J; Das, K; Dharia, C; Clark, Jr., A; Ryan, K; Hickey, M; et al.

    2009-01-01

    Novel inhibitors are needed to counteract the rapid emergence of drug-resistant HIV variants. HIV-1 reverse transcriptase (RT) has both DNA polymerase and RNase H (RNH) enzymatic activities, but approved drugs that inhibit RT target the polymerase. Inhibitors that act against new targets, such as RNH, should be effective against all of the current drug-resistant variants. Here, we present 2.80 {angstrom} and 2.04 {angstrom} resolution crystal structures of an RNH inhibitor, {beta}-thujaplicinol, bound at the RNH active site of both HIV-1 RT and an isolated RNH domain. {beta}-thujaplicinol chelates two divalent metal ions at the RNH active site. We provide biochemical evidence that {beta}-thujaplicinol is a slow-binding RNH inhibitor with noncompetitive kinetics and suggest that it forms a tropylium ion that interacts favorably with RT and the RNA:DNA substrate.

  12. Mauritia flexuosa Presents In Vitro and In Vivo Antiplatelet and Antithrombotic Activities.

    PubMed

    Fuentes, Eduardo; Rodríguez-Pérez, Wilson; Guzmán, Luis; Alarcón, Marcelo; Navarrete, Simón; Forero-Doria, Oscar; Palomo, Iván

    2013-01-01

    Fruit from the palm Mauritia flexuosa is one of the most important species in Peru, Venezuela, Brazil, Colombia, Bolivia, and Guyana. The present study aimed to investigate the antiplatelet and antithrombotic activities of oil extracted from Mauritia flexuosa. The fatty acid contents were determined by gas chromatography-mass spectrometry. Oil extract of peel of Mauritia flexuosa was extracted by soxhlet extraction. The oil extract inhibited platelet secretion and aggregation induced by ADP, collagen, and TRAP-6 by a concentration-dependent way (0.1 to 1 mg/mL) without the participation of the adenylyl cyclase pathway and diminished platelet rolling and firm adhesion under flow conditions. Furthermore, the oil extract induced a marked increase in the rolling speed of leukocytes retained on the platelet surface, reflecting a reduction of rolling and less adhesion. At the concentrations used, the oil extract significantly decreased platelet release of sP-selectin, an atherosclerotic-related inflammatory mediator. Oil extract inhibited thrombus growth at the same concentration as that of aspirin, a classical reference drug. Finally, the data presented herein also demonstrate for the first time to our knowledge the protective effect of oil extracted from Mauritia flexuosa on platelet activation and thrombosis formation.

  13. OSCE and Case Presentations As Active Assessments of Dental Student Performance.

    PubMed

    Park, Sang E; Anderson, Nina K; Karimbux, Nadeem Y

    2016-03-01

    The aim of this study was to evaluate whether the objective structured clinical examination (OSCE) and case presentation (CP) as forms of active assessment were effective measures of overall didactic knowledge and clinical performance in a predoctoral dental curriculum. This evaluation was conducted by statistical analysis of quality points (QP) awarded for didactic and clinical performance, CP grades, and OSCE scores for 185 students at Harvard School of Dental Medicine who graduated during the period 2010-14. As part of the requirements for graduation, each student takes three OSCEs and presents two patient cases. Data for the study were obtained from the Office of the Registrar. The results showed no direct correlation between QP and CP grades and no correlation between CP grades and OSCE scores. However, there was a correlation between OSCE scores and QP. Students with honors-level scores on any of the three OSCEs received significantly more QP than students who did not receive honors. In addition, students with passing scores on OSCEs 2 and 3 received significantly more QP than students with failing or marginal OSCE scores. Innovative formats of active assessment such as OSCEs and CPs can promote a student-centered learning environment. These data indicated that, within this study population, there was a positive association between OSCE scores and clinical and didactic performance, supporting the value of OSCEs as a means of assessment. PMID:26933109

  14. Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells

    PubMed Central

    Vela Ramirez, J.E.; Roychoudhury, R.; Habte, H.H.; Cho, M. W.; Pohl, N. L. B.; Narasimhan, B.

    2015-01-01

    The functionalization of polymeric nanoparticles with ligands that target specific receptors on immune cells offers the opportunity to tailor adjuvant properties by conferring pathogen mimicking attributes to the particles. Polyanhydride nanoparticles are promising vaccine adjuvants with desirable characteristics such as immunomodulation, sustained antigen release, activation of antigen presenting cells, and stabilization of protein antigens. These capabilities can be exploited to design nanovaccines against viral pathogens, such as HIV-1, due to the important role of dendritic cells and macrophages in viral spread. In this work, an optimized process was developed for carbohydrate functionalization of HIV-1 antigen-loaded polyanhydride nanoparticles. The carbohydrate-functionalized nanoparticles preserved antigenic properties upon release and also enabled sustained antigen release kinetics. Particle internalization was observed to be chemistry-dependent with positively charged nanoparticles being taken up more efficiently by dendritic cells. Up-regulation of the activation makers CD40 and CD206 was demonstrated with carboxymethyl-α-d-mannopyranosyl-(1,2)-d-mannopyranoside functionalized nanoparticles. The secretion of the cytokines IL-6 and TNF-α was shown to be chemistry-dependent upon stimulation with carbohydrate-functionalized nanoparticles. These results offer important new insights upon the interactions between carbohydrate-functionalized nanoparticles and antigen presenting cells and provide foundational information for the rational design of targeted nanovaccines against HIV-1. PMID:25068589

  15. Mauritia flexuosa Presents In Vitro and In Vivo Antiplatelet and Antithrombotic Activities

    PubMed Central

    Fuentes, Eduardo; Rodríguez-Pérez, Wilson; Guzmán, Luis; Alarcón, Marcelo; Navarrete, Simón; Forero-Doria, Oscar; Palomo, Iván

    2013-01-01

    Fruit from the palm Mauritia flexuosa is one of the most important species in Peru, Venezuela, Brazil, Colombia, Bolivia, and Guyana. The present study aimed to investigate the antiplatelet and antithrombotic activities of oil extracted from Mauritia flexuosa. The fatty acid contents were determined by gas chromatography—mass spectrometry. Oil extract of peel of Mauritia flexuosa was extracted by soxhlet extraction. The oil extract inhibited platelet secretion and aggregation induced by ADP, collagen, and TRAP-6 by a concentration-dependent way (0.1 to 1 mg/mL) without the participation of the adenylyl cyclase pathway and diminished platelet rolling and firm adhesion under flow conditions. Furthermore, the oil extract induced a marked increase in the rolling speed of leukocytes retained on the platelet surface, reflecting a reduction of rolling and less adhesion. At the concentrations used, the oil extract significantly decreased platelet release of sP-selectin, an atherosclerotic-related inflammatory mediator. Oil extract inhibited thrombus growth at the same concentration as that of aspirin, a classical reference drug. Finally, the data presented herein also demonstrate for the first time to our knowledge the protective effect of oil extracted from Mauritia flexuosa on platelet activation and thrombosis formation. PMID:24454503

  16. Dissociating the past from the present in the activity of place cells.

    PubMed

    de Hoz, Livia; Wood, Emma R

    2006-01-01

    It has been proposed that declarative memories can be dependent on both an episodic and a semantic memory system. While the semantic system deals with factual information devoid of reference to its acquisition, the episodic system, characterized by mental time travel, deals with the unique past experience in which an event took place. Episodic memory is characteristically hippocampus-dependent. Place cells are recorded from the hippocampus of rodents and their firing reflects many of the key characteristics of episodic memory. For example, they encode information about "what" happens "where," as well as temporal information. However, when these features are expressed during an animal's behavior, the neuronal activity could merely be categorizing the present situation and could therefore reflect semantic memory rather than episodic memory. We propose that mental time travel is the key feature of episodic memory and that it should take a form, in the awake animal, similar to the replay of behavioral patterns of activity that has been observed in hippocampus during sleep. Using tasks designed to evoke episodic memory, one should be able to see memory reactivation of behaviorally relevant sequences of activity in the awake animal while recording from hippocampus and other cortical structures.

  17. Particle shape dependence of CD8+ T cell activation by artificial antigen presenting cells.

    PubMed

    Sunshine, Joel C; Perica, Karlo; Schneck, Jonathan P; Green, Jordan J

    2014-01-01

    Previous work developing particle-based acellular, artificial antigen presenting cells (aAPCs) has focused exclusively on spherical platforms. To explore the role of shape, we generated ellipsoidal PLGA microparticles with varying aspect ratios (ARs) and synthesized aAPCs from them. The ellipsoidal biomimetic aAPCs with high-AR showed significantly enhanced in vitro and in vivo activity above spherical aAPCs with particle volume and antigen content held constant. Confocal imaging indicates that CD8+ T cells preferentially migrate to and are activated by interaction with the long axis of the aAPC. Importantly, enhanced activity of high-AR aAPCs was seen in a mouse melanoma model, with high-AR aAPCs improving melanoma survival compared to non-cognate aAPCs (p = 0.004) and cognate spherical aAPCs (p = 0.05). These findings indicate that particle geometry is a critical design criterion in the generation of aAPCs, and may offer insight into the essential role of geometry in the interaction between CD8+ T cells and biological APCs. PMID:24099710

  18. European Space Agency detector development for space science: present and future activities

    NASA Astrophysics Data System (ADS)

    Duvet, L.; Bavdaz, M.; Crouzet, P. E.; Nelms, N.; Nowicki-Bringuier, Y. R.; Shortt, B.; Verhoeve, P.

    2014-07-01

    We report on the present and future detector development activities for the European Space Agency Science Programme. The development of European technology in that field is a key mission enabler for the program, which requires TRL6 (ISO scale) by end of the definition phase, so called "mission adoption". This is particularly true for Astronomy and fundamental physics type missions. Current activities are in particular targeting large format and p-channel CCD, NIR and MWIR, LWIR wavelength ranges as well as related ASIC controller. For the longer term future mission plan (so called M4, M5 and L2 missions, M3 being PLATO and L1 JUICE), the extreme ends of the spectrum will be addressed. An overview of the detector status for the Earth Observation program is given in appendix, as most of the technologies are directly applicable to some extent to science missions, in particular for Planetary missions. The specific validation activities in place in the future mission preparation office in support to the space science program will be eventually briefly detailed.

  19. Past and present active sedimentation and tectonics in the South Alboran Sea

    NASA Astrophysics Data System (ADS)

    d'Acremont, E.; Gorini, C.; El Abbassi, M.; Farran, M.; Leroy, S.; Mercier de Lépinay, B.; Migeon, S.; Poort, J.; Ammar, A.; Smit, J.; Do Couto, D.; Ercilla, G.; Alonso, B.

    2012-04-01

    Since the Tortonian, the thinned continental crust and the overlying sedimentary cover of the Alboran Sea are submitted to tectonic inversion due to the convergence between Eurasia and Africa. The past and present deformation is significant along the Moroccan margin where the MARLBORO-1 cruise in 2011, acquired 1100 km of mid-resolution seismic reflection along 20 profiles perpendicular and parallel to the margin, off Al Hoceima, to latitude 36°N. The study area located on the Xauen/Tofino banks and the South Alboran ridge off Morocco, shows signs of both past and present strong tectonic deformation, mass-movement deposits (mostly slides and mass flow deposits), and contourites. The lateral and longitudinal evolution of contourites and mass movement deposits and the geometric relationships between those deposits and active tectonic structures have been studied. In the distal margin, contourites and gravitational instabilities are the depositional systems that best record the tectonic signal of the area since at least the Messinian. On the two flanks of the Xauen/Tofino and South Alboran ridge, the sedimentary register affected by growth-faults is mainly composed of contourites. Internal strata pattern, spatial and temporal distribution of thickness and depocenters, and discontinuities help to infer sedimentary processes and their interaction with tectonics. In the southern Alboran Sea where the bathymetry shows abrupt slopes, the recurrent seismic activity seems to be the main factor triggering mass wasting as witnessed by the Mass transport complexes (MTCs). Recent MTCs originate from escarpments on the edge of the contourites. However, in most cases the seismic reflection data show the depositional bodies of numerous slides linked to the activity of growth-faults and thrusts observed on the Xauen and Tofino Bank's north flanks. Tectonic inversion is recorded since the late Miocene with an acceleration of the uplift and compressional activity evidenced during

  20. Target site mutation and reduced translocation are present in a glyphosate-resistant Lolium multiflorum Lam. biotype from Spain.

    PubMed

    González-Torralva, Fidel; Gil-Humanes, Javier; Barro, Francisco; Brants, Ivo; De Prado, Rafael

    2012-09-01

    The resistance mechanism of a glyphosate-resistant Lolium multiflorum Lam. biotype collected in Córdoba (Southern Spain) was examined. Resistance Factor values at three different growth stages ranged between 4.77 and 4.91. At 96 hours after treatment (HAT) the S biotype had accumulated seven times more shikimic acid than the R biotype. There were significant differences in translocation of (14)C-glyphosate between biotypes, i.e. at 96 HAT, the R biotype accumulated in the treated leaf more than 70% of the absorbed herbicide, in comparison with 59.21% of the S biotype; the R biotype translocated only 14.79% of the absorbed (14)C-glyphosate to roots, while in the S population this value was 24.79%. Visualization of (14)C-glyphosate by phosphor imaging showed a reduced distribution in the R biotype compared with the S. Glyphosate metabolism was not involved in the resistance mechanism due to both biotypes showing similar values of glyphosate at 96 HAT. Comparison of the EPSPS gene sequences between biotypes indicated that the R biotype has a proline 182 to serine amino acid substitution. In short, the resistance mechanism of the L. multiflorum Lam. biotype is due to an impaired translocation of the herbicide and an altered target site.

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

    SciTech Connect

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

    2009-01-01

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

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

    SciTech Connect

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong; Chen, Shoudeng; Zhou, Jie; He, Xiaojing; Lv, Zongyang; Ge, Ruowen; Li, Xuemei; Deng, Tao; Fodor, Ervin; Rao, Zihe; Liu, Yingfang

    2009-11-10

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

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

    PubMed

    Silva, Pedro J; Rodrigues, Viviana

    2015-01-01

    Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H(+)-assisted cleavage of the Hg-C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg(2+) from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol(-1), determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after

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

    PubMed Central

    Rodrigues, Viviana

    2015-01-01

    Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H+-assisted cleavage of the Hg–C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg2+ from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol−1, determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after

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

    PubMed

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

    2003-01-01

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

  6. The modality-switch effect: visually and aurally presented prime sentences activate our senses

    PubMed Central

    Scerrati, Elisa; Baroni, Giulia; Borghi, Anna M.; Galatolo, Renata; Lugli, Luisa; Nicoletti, Roberto

    2015-01-01

    Verifying different sensory modality properties for concepts results in a processing cost known as the modality-switch effect. It has been argued that this cognitive cost is the result of a perceptual simulation. This paper extends this argument and reports an experiment investigating whether the effect is the result of an activation of sensory information which can also be triggered by perceptual linguistically described stimuli. Participants were first exposed to a prime sentence describing a light or a sound’s perceptual property (e.g., “The light is flickering”, “The sound is echoing”), then required to perform a property-verification task on a target sentence (e.g., “Butter is yellowish”, “Leaves rustle”). The content modalities of the prime and target sentences could be compatible (i.e., in the same modality: e.g., visual–visual) or not (i.e., in different modalities). Crucially, we manipulated the stimuli’s presentation modality such that half of the participants was faced with written sentences while the other half was faced with aurally presented sentences. Results show a cost when two different modalities alternate, compared to when the same modality is repeated with both visual and aural stimuli presentations. This result supports the embodied and grounded cognition view which claims that conceptual knowledge is grounded into the perceptual system. Specifically, this evidence suggests that sensory modalities can be pre-activated through the simulation of either read or listened linguistic stimuli describing visual or acoustic perceptual properties. PMID:26579049

  7. Xenohormetic and anti-aging activity of secoiridoid polyphenols present in extra virgin olive oil

    PubMed Central

    Menendez, Javier A.; Joven, Jorge; Aragonès, Gerard; Barrajón-Catalán, Enrique; Beltrán-Debón, Raúl; Borrás-Linares, Isabel; Camps, Jordi; Corominas-Faja, Bruna; Cufí, Sílvia; Fernández-Arroyo, Salvador; Garcia-Heredia, Anabel; Hernández-Aguilera, Anna; Herranz-López, María; Jiménez-Sánchez, Cecilia; López-Bonet, Eugeni; Lozano-Sánchez, Jesús; Luciano-Mateo, Fedra; Martin-Castillo, Begoña; Martin-Paredero, Vicente; Pérez-Sánchez, Almudena; Oliveras-Ferraros, Cristina; Riera-Borrull, Marta; Rodríguez-Gallego, Esther; Quirantes-Piné, Rosa; Rull, Anna; Tomás-Menor, Laura; Vazquez-Martin, Alejandro; Alonso-Villaverde, Carlos; Micol, Vicente; Segura-Carretero, Antonio

    2013-01-01

    Aging can be viewed as a quasi-programmed phenomenon driven by the overactivation of the nutrient-sensing mTOR gerogene. mTOR-driven aging can be triggered or accelerated by a decline or loss of responsiveness to activation of the energy-sensing protein AMPK, a critical gerosuppressor of mTOR. The occurrence of age-related diseases, therefore, reflects the synergistic interaction between our evolutionary path to sedentarism, which chronically increases a number of mTOR activating gero-promoters (e.g., food, growth factors, cytokines and insulin) and the “defective design” of central metabolic integrators such as mTOR and AMPK. Our laboratories at the Bioactive Food Component Platform in Spain have initiated a systematic approach to molecularly elucidate and clinically explore whether the “xenohormesis hypothesis,” which states that stress-induced synthesis of plant polyphenols and many other phytochemicals provides an environmental chemical signature that upregulates stress-resistance pathways in plant consumers, can be explained in terms of the reactivity of the AMPK/mTOR-axis to so-called xenohormetins. Here, we explore the AMPK/mTOR-xenohormetic nature of complex polyphenols naturally present in extra virgin olive oil (EVOO), a pivotal component of the Mediterranean style diet that has been repeatedly associated with a reduction in age-related morbid conditions and longer life expectancy. Using crude EVOO phenolic extracts highly enriched in the secoiridoids oleuropein aglycon and decarboxymethyl oleuropein aglycon, we show for the first time that (1) the anticancer activity of EVOO secoiridoids is related to the activation of anti-aging/cellular stress-like gene signatures, including endoplasmic reticulum (ER) stress and the unfolded protein response, spermidine and polyamine metabolism, sirtuin-1 (SIRT1) and NRF2 signaling; (2) EVOO secoiridoids activate AMPK and suppress crucial genes involved in the Warburg effect and the self-renewal capacity of

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

    PubMed

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

    2014-03-01

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

  9. Effective Presentation of Metabolic Rate Information for Lunar Extravehicular Activity (EVA)

    NASA Technical Reports Server (NTRS)

    Mackin, Michael A.; Gonia, Philip; Lombay-Gonzalez, Jose

    2010-01-01

    During human exploration of the lunar surface, a suited crewmember needs effective and accurate information about consumable levels remaining in their life support system. The information must be presented in a manner that supports real-time consumable monitoring and route planning. Since consumable usage is closely tied to metabolic rate, the lunar suit must estimate metabolic rate from life support sensors, such as oxygen tank pressures, carbon dioxide partial pressure, and cooling water inlet and outlet temperatures. To provide adequate warnings that account for traverse time for a crewmember to return to a safe haven, accurate forecasts of consumable depletion rates are required. The forecasts must be presented to the crewmember in a straightforward, effective manner. In order to evaluate methods for displaying consumable forecasts, a desktop-based simulation of a lunar Extravehicular Activity (EVA) has been developed for the Constellation lunar suite s life-support system. The program was used to compare the effectiveness of several different data presentation methods.

  10. Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure

    PubMed Central

    O'Farrell, Paul A.; Joshua-Tor, Leemor

    2006-01-01

    Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes. PMID:17007609

  11. Transforming growth factor. beta. sub 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis

    SciTech Connect

    Broekelmann, T.J.; Limper, A.H.; McDonald, J.A. ); Colby, T.V. )

    1991-08-01

    Idiopathic pulmonary fibrosis is an inexorably fatal disorder characterized by connective tissue deposition within the terminal air spaces resulting in loss of lung function and eventual respiratory failure. Previously, the authors demonstrated that foci of activated fibroblasts expressing high levels of fibronectin, procollagen, and smooth muscle actin and thus resembling those found in healing wounds are responsible for the connective tissue deposition and scarring in idiopathic pulmonary fibrosis. Using in situ hybridization and immunohistochemistry, they now demonstrate the presence of transforming growth factor {beta}{sub 1} (TGF-{beta}{sub 1}), a potent profibrotic cytokine, in the foci containing these activated fibroblasts. These results suggest that matrix-associated TGF-{beta}{sub 1} may serve as a stimulus for the persistent expression of connective tissue genes. One potential source of the TGF-{beta}{sub 1} is the alveolar macrophage, and they demonstrate the expression of abundant TGF-{beta}{sub 1} mRNA in alveolar macrophages in lung tissue from patients with idiopathic pulmonary fibrosis.

  12. 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.

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

    SciTech Connect

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

    2015-06-04

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

  14. A local reaction at or near injection site: case definition and guidelines for collection, analysis, and presentation of immunization safety data.

    PubMed

    Gidudu, Jane; Kohl, Katrin S; Halperin, Scott; Hammer, Sandra Jo; Heath, Paul T; Hennig, Renald; Hoet, Bernard; Rothstein, Edward; Schuind, Anne; Varricchio, Frederick; Walop, Wikke

    2008-12-01

    The need for developing a case definition and guidelines for a local reaction at or near the injection site, methods for the development of the case definition and guidelines as an adverse event following immunization as well as the rationale for selected decisions about the case definition for a local reaction at or near the injection site are explained in the Preamble section. The case definition is structured in 2 levels of diagnostic certainty: level 1 includes any description of morphological or physiological change at or near the injection site that is described or identified by a healthcare provider. Level 2 is any description of morphological or physiological change at or near injection site that is described by any other person. In Guidelines section, the working group recommends to enable meaningful and standardized data collection, analysis, and presentation of information about a local reaction at or near the injection site. However, implementation of all guidelines might not be possible in all settings. The availability of information may vary depending upon resources, geographic region, and whether the source of information is a prospectively designed clinical trial, a post-marketing surveillance or epidemiologic study, or an individual report of a local reaction at injection site.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Fenwick, Michael K; Mehta, Angad P; Zhang, Yang; Abdelwahed, Sameh H; Begley, Tadhg P; Ealick, Steven E

    2015-03-27

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

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

    SciTech Connect

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

    2009-04-29

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

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

    SciTech Connect

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-27

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

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

    PubMed

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

    2001-08-01

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

  20. Selective Sirt2 inhibition by ligand-induced rearrangement of the active site.

    PubMed

    Rumpf, Tobias; Schiedel, Matthias; Karaman, Berin; Roessler, Claudia; North, Brian J; Lehotzky, Attila; Oláh, Judit; Ladwein, Kathrin I; Schmidtkunz, Karin; Gajer, Markus; Pannek, Martin; Steegborn, Clemens; Sinclair, David A; Gerhardt, Stefan; Ovádi, Judit; Schutkowski, Mike; Sippl, Wolfgang; Einsle, Oliver; Jung, Manfred

    2015-01-01

    Sirtuins are a highly conserved class of NAD(+)-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology. PMID:25672491

  1. Selective Sirt2 inhibition by ligand-induced rearrangement of the active site

    PubMed Central

    Rumpf, Tobias; Schiedel, Matthias; Karaman, Berin; Roessler, Claudia; North, Brian J.; Lehotzky, Attila; Oláh, Judit; Ladwein, Kathrin I.; Schmidtkunz, Karin; Gajer, Markus; Pannek, Martin; Steegborn, Clemens; Sinclair, David A.; Gerhardt, Stefan; Ovádi, Judit; Schutkowski, Mike; Sippl, Wolfgang; Einsle, Oliver; Jung, Manfred

    2015-01-01

    Sirtuins are a highly conserved class of NAD+-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology. PMID:25672491

  2. Synthetic models of the active site of catechol oxidase: mechanistic studies.

    PubMed

    Koval, Iryna A; Gamez, Patrick; Belle, Catherine; Selmeczi, Katalin; Reedijk, Jan

    2006-09-01

    The ability of copper proteins to process dioxygen at ambient conditions has inspired numerous research groups to study their structural, spectroscopic and catalytic properties. Catechol oxidase is a type-3 copper enzyme usually encountered in plant tissues and in some insects and crustaceans. It catalyzes the conversion of a large number of catechols into the respective o-benzoquinones, which subsequently auto-polymerize, resulting in the formation of melanin, a dark pigment thought to protect a damaged tissue from pathogens. After the report of the X-ray crystal structure of catechol oxidase a few years earlier, a large number of publications devoted to the biomimetic modeling of its active site appeared in the literature. This critical review (citing 114 references) extensively discusses the synthetic models of this enzyme, with a particular emphasis on the different approaches used in the literature to study the mechanism of the catalytic oxidation of the substrate (catechol) by these compounds. These are the studies on the substrate binding to the model complexes, the structure-activity relationship, the kinetic studies of the catalytic oxidation of the substrate and finally the substrate interaction with (per)oxo-dicopper adducts. The general overview of the recognized types of copper proteins and the detailed description of the crystal structure of catechol oxidase, as well as the proposed mechanisms of the enzymatic cycle are also presented. PMID:16936929

  3. New Isotopic Constraints on the Sources of Methane at Sites of Active Continental Serpentinization

    NASA Astrophysics Data System (ADS)

    Wang, D. T.; Gruen, D.; Morrill, P. L.; Rietze, A.; Nealson, K. H.; Kubo, M. D.; Cardace, D.; Schrenk, M. O.; Hoehler, T. M.; McCollom, T. M.; Etiope, G.; Hosgormez, H.; Schoell, M.; Ono, S.

    2014-12-01

    At continental sites of serpentinization, high concentrations of reduced gases (e.g., H2, CH4) are frequently found in association with highly-alkaline groundwater. Identification of the process(es) responsible for the generation of methane—as well as the source(s) of C & H—in these environments has been challenging. The difficulty is due to both the wide range of processes (microbial, thermal, abiotic) that could be involved, and the limited number of parameters that are accessible to currently-available analytical technologies (e.g., δ13C, δD). The recent development of a new technique based on tunable infrared laser spectroscopy [1] has enabled the fully-resolved quantification of four isotopologues of methane: 12CH4, 13CH4, 12CH3D, and 13CH3D, a doubly-substituted ("clumped") isotopologue. We used this technique to measure 13CH3D in gases sampled from continental sites of serpentinization, in order to provide independent constraints on C-H bond-forming processes involved in the generation of the methane found in these systems. Our study sites are hosted in ultramafic units that are presently undergoing serpentinization. These include The Cedars peridotite body (Calif., USA) [2], the Coast Range Ophiolite Microbial Observatory (Calif., USA) [3], and the Chimaera seep (Tekirova Ophiolite, Turkey) [4]. Preliminary measurements indicate that Δ13CH3D (the deviation of the abundance of 13CH3D from the stochastic distribution) in methane sampled from these sites spans nearly the entire range of thermodynamically-predicted values, from >+5‰ (13CH3D-based apparent equilibrium temperature < 45 °C) to ~0‰ (Tapparent → ∞). The new 13CH3D data is complemented by conventional geochemical analyses (e.g., dissolved ions/organics, δ13C, δD) on samples collected during the same field campaigns. Our study demonstrates that the measurement of 13CH3D provides a new dimension of isotopic constraints for unraveling the complex processes controlling the distribution

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

    PubMed Central

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

    2015-01-01

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

  5. Characterizations of Metal Binding in the Active Sites of Acireductone Dioxygenase Isoforms from Klebsiella ATCC 8724

    SciTech Connect

    Chai,S.; Ju, T.; Dang, M.; Goldsmith, R.; Maroney, M.; Pochapsky, T.

    2008-01-01

    The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities toward their common substrates (1, 2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M2+ metal ion bound in the active site. The Ni2+-bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate, and carbon monoxide, while the Fe2+-bound FeARD' catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS), and isothermal calorimetry measurements, it is concluded that the same four residues, His96, His98, Glu102 and His140, provide the protein-based ligands for the metal in both the Ni- and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD' and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a histidine ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved nonligand acidic residues, Glu95 and Glu100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu100 mutant reconstitutes readily but has low activity. Mutation of Asp101 results in an active enzyme that incorporates metal in vivo but

  6. Characterization of Metal Binding in the Active Sites of acireductone dioxygenase Isoforms from Klebsiella ATCC 8724

    SciTech Connect

    S Chai; T Ju; M Dang; R Goldsmith; M Maroney; T Pochapsky

    2011-12-31

    The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities toward their common substrates (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M{sup 2+} metal ion bound in the active site. The Ni{sup 2+}-bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate, and carbon monoxide, while the Fe{sup 2+}-bound FeARD catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS), and isothermal calorimetry measurements, it is concluded that the same four residues, His96, His98, Glu102 and His140, provide the protein-based ligands for the metal in both the Ni- and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a histidine ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved nonligand acidic residues, Glu95 and Glu100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu100 mutant reconstitutes readily but has low activity. Mutation of Asp101 results in an active enzyme that incorporates

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

    PubMed

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

    2016-07-01

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

  8. Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase V by structural and functional studies.

    PubMed

    Rajan, Rakhi; Prasad, Rajendra; Taneja, Bhupesh; Wilson, Samuel H; Mondragón, Alfonso

    2013-01-01

    Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix-hairpin-helix [(HhH)(2)] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)(2) domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo. PMID:23125368

  9. Spacing between GT-1 binding sites within a light-responsive element is critical for transcriptional activity.

    PubMed Central

    Gilmartin, P M; Chua, N H

    1990-01-01

    Dissection of the light-responsive element (LRE) located between -166 and -50 of rbcS-3A from pea has revealed critical spacing requirements between the two GT-1 binding sites for light-responsive transcription. An increase in spacing between the two sites by as little as 2 bp reduces dramatically the rbcS-3A transcript levels in vivo. Mutation of the 10 bp between the binding sites leads to slightly lower transcript levels, as do deletions of either 3 bp or 8 bp. Deletions of 5 bp or 7 bp from between the GT-1 binding sites do not affect rbcS-3A transcript levels; however, a deletion of 10 bp virtually abolishes the activity of this element. These spacing changes within the light-responsive element similarly affect transcription of a divergently oriented and truncated nopaline synthase promoter. Most spacing changes between the two GT-1 binding sites, however, do not impair the binding of GT-1 to this element in vitro. Together with previous observations, these results suggest that the nuclear factor GT-1 may interact with the binding sites in either a productive or nonproductive manner and that GT-1 binding is necessary but not sufficient for light-responsive transcription. We also discuss our results in relation to the observed spacing of similar sequence elements present within other light-responsive promoters. PMID:2152170

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

    SciTech Connect

    Fitzner, R.E.; Weiss, S.G.; Stegen, J.A.

    1994-06-01

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

  11. Fighting the stigma caused by mental disorders: past perspectives, present activities, and future directions

    PubMed Central

    STUART, HEATHER

    2008-01-01

    People who live with mental illnesses are among the most stigmatized groups in society. In 1996, in recognition of the particularly harsh burden caused by the stigma associated with schizophrenia, the WPA initiated a global anti-stigma program, Open-the-Doors. In 2005, a WPA Section on Stigma and Mental Health was created, with a broader mandate to reduce stigma and discrimination caused by mental disabilities in general. In light of these impor-tant developments, and the growing public health interest in stigma reduction, this paper reflects on the past perspectives that have led us to our current position, reviews present activities and accomplishments, and identifies challenges that the Section members will face in their future efforts to reduce the stigma caused by mental disorders. PMID:18836546

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

    PubMed

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

    2016-08-18

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  14. A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

    SciTech Connect

    Somarowthu, Srinivas; Brodkin, Heather R.; D’Aquino, J. Alejandro; Ringe, Dagmar; Ondrechen, Mary Jo; Beuning, Penny J.

    2012-07-11

    Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

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

    SciTech Connect

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

    1986-04-01

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

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

    PubMed

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

    2015-12-15

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

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

    PubMed Central

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

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr890, Ser893, Thr894 and Thr899, differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  18. 21st Century Extravehicular Activities: Synergizing Past and Present Training Methods for Future Spacewalking Success

    NASA Technical Reports Server (NTRS)

    Moore, Sandra K.; Gast, Matthew A.

    2009-01-01

    Neil Armstrong's understated words, "That's one small step for man, one giant leap for mankind." were spoken from Tranquility Base forty years ago. Even today, those words resonate in the ears of millions, including many who had yet to be born when man first landed on the surface of the moon. By their very nature, and in the the spirit of exploration, extravehicular activities (EVAs) have generated much excitement throughout the history of manned spaceflight. From Ed White's first space walk in June of 1965, to the first steps on the moon in 1969, to the expected completion of the International Space Station (ISS), the ability to exist, live and work in the vacuum of space has stood as a beacon of what is possible. It was NASA's first spacewalk that taught engineers on the ground the valuable lesson that successful spacewalking requires a unique set of learned skills. That lesson sparked extensive efforts to develop and define the training requirements necessary to ensure success. As focus shifted from orbital activities to lunar surface activities, the required skill-set and subsequently the training methods, changed. The requirements duly changed again when NASA left the moon for the last time in 1972 and have continued to evolve through the Skylab, Space Shuttle; and ISS eras. Yet because the visits to the moon were so long ago, NASA's expertise in the realm of extra-terrestrial EVAs has diminished. As manned spaceflight again shifts its focus beyond low earth orbit, EVA success will depend on the ability to synergize the knowledge gained over 40+ years of spacewalking to create a training method that allows a single crewmember to perform equally well, whether performing an EVA on the surface of the Moon, while in the vacuum of space, or heading for a rendezvous with Mars. This paper reviews NASA's past and present EVA training methods and extrapolates techniques from both to construct the basis for future EVA astronaut training.

  19. 21st Century extravehicular activities: Synergizing past and present training methods for future spacewalking success

    NASA Astrophysics Data System (ADS)

    Moore, Sandra K.; Gast, Matthew A.

    2010-10-01

    Neil Armstrong's understated words, "That's one small step for man, one giant leap for mankind" were spoken from Tranquility Base forty years ago. Even today, those words resonate in the ears of millions, including many who had yet to be born when man first landed on the surface of the moon. By their very nature, and in the true spirit of exploration, extravehicular activities (EVAs) have generated much excitement throughout the history of manned spaceflight. From Ed White's first spacewalk in the June of 1965, to the first steps on the moon in 1969, to the expected completion of the International Space Station (ISS), the ability to exist, live and work in the vacuum of space has stood as a beacon of what is possible. It was NASA's first spacewalk that taught engineers on the ground the valuable lesson that successful spacewalking requires a unique set of learned skills. That lesson sparked extensive efforts to develop and define the training requirements necessary to ensure success. As focus shifted from orbital activities to lunar surface activities, the required skill set and subsequently the training methods changed. The requirements duly changed again when NASA left the moon for the last time in 1972 and have continued to evolve through the SkyLab, Space Shuttle, and ISS eras. Yet because the visits to the moon were so long ago, NASA's expertise in the realm of extra-terrestrial EVAs has diminished. As manned spaceflight again shifts its focus beyond low earth orbit, EVA's success will depend on the ability to synergize the knowledge gained over 40+ years of spacewalking to create a training method that allows a single crewmember to perform equally well, whether performing an EVA on the surface of the Moon, while in the vacuum of space, or heading for a rendezvous with Mars. This paper reviews NASA's past and present EVA training methods and extrapolates techniques from both to construct the basis for future EVA astronaut training.

  20. Deep-Sea Magnetics on Active and Fossil Hydrothermal Sites: a Tool to Detect and Characterize Submarine Ore Deposits

    NASA Astrophysics Data System (ADS)

    Dyment, J.; Szitkar, F.; Fouquet, Y.; Choi, Y.

    2011-12-01

    /V Pourquoi pas?, 2007) and MoMARDream (R/V L'Atalante, 2008), both using ROV Victor of IFREMER, on basalt and ultramafic-hosted sites of the Mid-Atlantic Ridge, will be presented. High resolution, deep-sea magnetic investigations may therefore significantly contribute to the detection and characterization of active and fossil sites and to the evaluation of their mining potential, for instance through a fleet of small AUV exploring systematically the deep seafloor.

  1. Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells.

    PubMed

    Vela Ramirez, J E; Roychoudhury, R; Habte, H H; Cho, M W; Pohl, N L B; Narasimhan, B

    2014-01-01

    The functionalization of polymeric nanoparticles with ligands that target specific receptors on immune cells offers the opportunity to tailor adjuvant properties by conferring pathogen mimicking attributes to the particles. Polyanhydride nanoparticles are promising vaccine adjuvants with desirable characteristics such as immunomodulation, sustained antigen release, activation of antigen presenting cells (APCs), and stabilization of protein antigens. These capabilities can be exploited to design nanovaccines against viral pathogens, such as HIV-1, due to the important role of dendritic cells (DCs) and macrophages in viral spread. In this work, an optimized process was developed for carbohydrate functionalization of HIV-1 antigen-loaded polyanhydride nanoparticles. The carbohydrate-functionalized nanoparticles preserved antigenic properties upon release and also enabled sustained antigen release kinetics. Particle internalization was observed to be chemistry-dependent with positively charged nanoparticles being taken up more efficiently by DCs. Up-regulation of the activation makers CD40 and CD206 was demonstrated with carboxymethyl-α-d-mannopyranosyl-(1,2)-d-mannopyranoside functionalized nanoparticles. The secretion of the cytokines IL-6 and TNF-α was shown to be chemistry-dependent upon stimulation with carbohydrate-functionalized nanoparticles. These results offer important new insights upon the interactions between carbohydrate-functionalized nanoparticles and APCs and provide foundational information for the rational design of targeted nanovaccines against HIV-1. PMID:25068589

  2. An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions.

    PubMed

    Sengupta, Raghuvir N; Van Schie, Sabine N S; Giambaşu, George; Dai, Qing; Yesselman, Joseph D; York, Darrin; Piccirilli, Joseph A; Herschlag, Daniel

    2016-01-01

    Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2'- and 3'-deoxy (-H) and -amino (-NH(2)) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3'-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2'-OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M(C), to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function.

  3. A summary of present-day gully formation and activity on Mars

    NASA Astrophysics Data System (ADS)

    Diniega, Serina; Hansen, Candice; McEwen, Alfred; Dundas, Colin; Byrne, Shane

    2016-07-01

    trends and variations in gully size, morphology, orientation, and location are additionally examined as indications of a seasonal (or other) control, as well as variations with latitude, dune type/size/orientation, or Mars year. This presentation will summarize the results of our monitoring campaigns, and will compare the data that are available about gullies of different morphologies and locations. Our aim is to identify possible similarities (and differences) in gully activity processes across Mars, and to estimate the rates of gully formation and modification processes (as has been done for gullies in the southern mid-latitudes [3]). Such estimates would aid interpretation of the likely ages of observed gullies, which have implications for whether records of past (and potential aqueous) gully formation processes are still observable. [1] Diniega et al. (2010) Geology, 38, 1047-1050. [2] Dundas et al. (2012) Icarus, 220, 124-143. [3] Dundas et al. (2015) Icarus, 251, 244-263. [4] Dundas et al. (2010) GRL, 37, L07202. [5] Vincendon (2015) JGR, 120, 1859-1879. [6] Harrison et al. (2015) Icarus, 252, 236-254. [7] Pilorget & Forget (2016) Nature Geosci. 9, 65-69. [8] Vincendon et al. (2010) JGR, 115, E10001. [9] Horgan & Bell (2012) Geophys. Res. Lett. 39, L09201. [10] Hansen et al., (2011) Science 331, 575-578. [11] Hansen et al. (2015) Icarus 251, 264-267.

  4. Wobble Pairs of the HDV Ribozyme Play Specific Roles in Stabilization of Active Site Dynamics

    PubMed Central

    Sripathi, Kamali N.; Banáš, Pavel; Reblova, Kamila; Šponer, Jiři; Otyepka, Michal

    2015-01-01

    The hepatitis delta virus (HDV) is the only known human pathogen whose genome contains a catalytic RNA motif (ribozyme). The overall architecture of the HDV ribozyme is that of a double-nested pseudoknot, with two GU pairs flanking the active site. Although extensive studies have shown that mutation of either wobble results in decreased catalytic activity, little work has focused on linking these mutations to specific structural effects on catalytic fitness. Here we use molecular dynamics simulations based on an activated structure to probe the active site dynamics as a result of wobble pair mutations. In both wild-type and mutant ribozymes, the in-line fitness of the active site (as a measure of catalytic proficiency) strongly depends on the presence of a C75(N3H3+)N1(O5′) hydrogen bond, which positions C75 as the general acid for the reaction. Our mutational analyses show that each GU wobble supports catalytically fit conformations in distinct ways; the reverse G25U20 wobble promotes high in-line fitness, high occupancy of the C75(N3H3+)G1(O5′) general-acid hydrogen bond and stabilization of the G1U37 wobble, while the G1U37 wobble acts more locally by stabilizing high in-line fitness and the C75(N3H3+)G1(O5′) hydrogen bond. We also find that stable type I A-minor and P1.1 hydrogen bonding above and below the active site, respectively, prevent local structural disorder from spreading and disrupting global conformation. Taken together, our results define specific, often redundant architectural roles for several structural motifs of the HDV ribozyme active site, expanding the known roles of these motifs within all HDV-like ribozymes and other structured RNAs. PMID:25631765

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

    PubMed Central

    Colombo, Matteo; Girard, Eric; Franzetti, Bruno

    2016-01-01

    TET aminopeptidases are dodecameric particles shared in the three life domains involved in various biological processes, from carbon source provider in archaea to eye-pressure regulation in humans. Each subunit contains a dinuclear metal site (M1 and M2) responsible for the enzyme catalytic activity. However, the role of each metal ion is still uncharacterized. Noteworthy, while mesophilic TETs are activated by Mn2+, hyperthermophilic TETs prefers Co2+. Here, by means of anomalous x-ray crystallography and enzyme kinetics measurements of the TET3 aminopeptidase from the hyperthermophilic organism Pyrococcus furiosus (PfTET3), we show that M2 hosts the catalytic activity of the enzyme, while M1 stabilizes the TET3 quaternary structure and controls the active site flexibility in a temperature dependent manner. A new third metal site (M3) was found in the substrate binding pocket, modulating the PfTET3 substrate preferences. These data show that TET activity is tuned by the molecular interplay among three metal sites. PMID:26853450

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

    NASA Astrophysics Data System (ADS)

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

    2013-05-01

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

  7. Active-Site Monovalent Cations Revealed in a 1.55 Å Resolution Hammerhead Ribozyme Structure

    PubMed Central

    Anderson, Michael; Schultz, Eric P.; Martick, Monika; Scott, William G.

    2013-01-01

    We have obtained a 1.55 Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni in conditions that permit detailed observations of Na+ ion binding in the ribozyme's active site. At least two such Na+ ions are observed. The first Na+ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical to that previously observed for divalent cations. A second Na+ ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na+, but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na+ directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest resolution ribozyme structure in the protein data bank. PMID:23711504

  8. (Function of active-site residues of ribulosebisphosphate carboxylase/oxygenase, Stockholm, Sweden, and visit to Uppsala, Sweden, August 6--12, 1989): Foreign trip report

    SciTech Connect

    Hartman, F.C.

    1989-08-22

    The traveler participated in the 8th International Congress on Photosynthesis by presenting a paper entitled ''Function of Active-Site Residues of Ribulosebisphosphate Carboxylase/Oxygenase'' and by chairing a discussion session on the same enzyme. Presentation concerning biological CO/sub 2/ fixation, chemical modifications of proteins, 3D structure of proteins, and site-directed mutagenesis were relevant to ongoing investigations of the Protein Engineering Program at ORNL's Biology Division.

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

    SciTech Connect

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

    1998-08-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

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

    PubMed Central

    Herter, Susanne; Kranz, David C; Turner, Nicholas J

    2015-01-01

    Summary Cytochrome P450 monooxygenases are useful biocatalysts for C–H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations. PMID:26664590

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

    PubMed

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

    2015-01-01

    Cytochrome P450 monooxygenases are useful biocatalysts for C-H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations.

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

    PubMed

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

    2015-01-01

    Cytochrome P450 monooxygenases are useful biocatalysts for C-H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations. PMID:26664590

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

    PubMed

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

    2007-08-14

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

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

    PubMed

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

    1999-02-19

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

  18. Methanol Synthesis over Cu/ZnO/Al2O3: The Active Site in Industrial Catalysis

    SciTech Connect

    Behrens, Malte

    2012-03-28

    Unlike homogeneous catalysts, heterogeneous catalysts that have been optimized through decades are typically so complex and hard to characterize that the nature of the catalytically active site is not known. This is one of the main stumbling blocks in developing rational catalyst design strategies in heterogeneous catalysis. We show here how to identify the crucial atomic structure motif for the industrial Cu/ZnO/Al{sub 2}O{sub 3} methanol synthesis catalyst. Using a combination of experimental evidence from bulk-, surface-sensitive and imaging methods collected on real high-performance catalytic systems in combination with DFT calculations. We show that the active site consists of Cu steps peppered with Zn atoms, all stabilized by a series of well defined bulk defects and surface species that need jointly to be present for the system to work.

  19. Active sites in Cu-SSZ-13 deNOx catalyst under reaction conditions: a XAS/XES perspective

    NASA Astrophysics Data System (ADS)

    Lomachenko, Kirill A.; Borfecchia, Elisa; Bordiga, Silvia; Soldatov, Alexander V.; Beato, Pablo; Lamberti, Carlo

    2016-05-01

    Cu-SSZ-13 is a highly active catalyst for the NH3-assisted selective catalytic reduction (SCR) of the harmful nitrogen oxides (NOx, x=1, 2). Since the catalytically active sites for this reaction are mainly represented by isolated Cu ions incorporated into the zeolitic framework, element-selective studies of Cu local environment are crucial to fully understand the enhanced catalytic properties of this material. Herein, we highlight the recent advances in the characterization of the most abundant Cu-sites in Cu-SSZ-13 upon different reaction-relevant conditions made employing XAS and XES spectroscopies, complemented by computational analysis. A concise review of the most relevant literature is also presented.

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

    PubMed

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

    2002-05-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2007-10-01

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

  3. Artificial antigen presenting cell (aAPC) mediated activation and expansion of natural killer T cells.

    PubMed

    East, James E; Sun, Wenji; Webb, Tonya J

    2012-01-01

    Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells(1). Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules(2). NKT cells express an invariant TCRα chain rearrangement: Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβ chains of limited diversity(3-6), and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d (7). The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with α-Galactosylceramide (α-GalCer) and a variety of cytokines(8). Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines(9,10). In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients(11). However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells(12, 13). Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is

  4. 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

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

    PubMed

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

    2003-02-19

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

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

    NASA Astrophysics Data System (ADS)

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

    1983-12-01

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

  7. 78 FR 18576 - Agency Information Collection Activities; Comment Request; Experimental Sites Data Collection...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-27

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

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

    PubMed Central

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

    2016-01-01

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

  9. The Thumbs Up Ecology Curriculum: A Fun Group of School Site Activities for Sixth Graders.

    ERIC Educational Resources Information Center

    Smith, John; And Others

    This guide is a collection of "fun" school site activities for sixth graders. Some of the topics covered are: animals, trees, energy and lifestyle, land use and you, energy conservation, and car-pooling. Each section offers both introductory information about the topic as well as questions to ponder such as what, so what, now what, and another way…

  10. 78 FR 33908 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-05

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

  11. 77 FR 3460 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-24

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

  12. 75 FR 71677 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-24

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

  13. Archaeological Activity Report: Post-Review Discoveries Within 45BN431 at Solid Waste Site 128-F-2

    SciTech Connect

    T. E. Marceau; J. J. Sharpe

    2006-12-21

    During monitoring of remedial activities at Solid Waste Site 128-F-2 on August 19, 2005, a concentration of mussel shell was discovered in the west wall of a trench in the northen section of the waste site.

  14. Glutamate-activated chloride channels: Unique fipronil targets present in insects but not in mammals

    PubMed Central

    Narahashi, Toshio; Zhao, Xilong; Ikeda, Tomoko; Salgado, Vincent L.; Yeh, Jay Z.

    2009-01-01

    Selectivity to insects over mammals is one of the important characteristics for a chemical to become a useful insecticide. Fipronil was found to block cockroach GABA receptors more potently than rat GABAA receptors. Furthermore, glutamate-activated chloride channels (GluCls), which are present in cockroaches but not in mammals, were very sensitive to the blocking action of fipronil. The IC50s of fipronil block were 30 nM in cockroach GABA receptors and 1600 nM in rat GABAA receptors. Moreover, GluCls of cockroach neurons had low IC50s for fipronil. Two types of glutamate-induced chloride current were obswerved: desensitizing and non-desensitizing, with fipronil IC50s of 800 and 10 nM, respectively. We have developed methods to separately record these two types of GluCls. The non-desensitizing and desensitizing currents were selectively inhibited by trypsin and polyvinylpyrrolidone, respectively. In conclusion, in addition to GABA receptors, GluCls play a crucial role in selectivity of fipronil to insects over mammals. GluCls form the basis for development of selective and safe insecticides. PMID:20563240

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

    PubMed

    Taylor, John C; Markham, George D

    2003-07-15

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

  16. Three Activities To Assist Biology Teachers in Presenting Conceptually Difficult Topics.

    ERIC Educational Resources Information Center

    Taylor, Neil; Tulip, David

    1997-01-01

    Outlines three activities for different areas of biology that can serve as motivators for students or as demonstrations. Each activity is easy to organize and uses available materials. Topics include evolution, anaerobic respiration, and heat loss. (DDR)

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

    PubMed

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

    2014-01-28

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

  18. Characterization of the active site properties of CYP4F12.

    PubMed

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

    2014-10-01

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

  19. Microbial Activity In The Peerless Jenny King Sulfate Reducing Bioreactor System (Presentation)

    EPA Science Inventory

    The Peerless Jenny King treatment system is a series of four sulfate reducing bioreactor cells installed to treat acid mine drainage in the Upper Tenmile Creek Superfund Site located in the Rimini Mining District, near Helena MT. The system consists of a wetland pretreatment fol...

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

    SciTech Connect

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

    1990-10-01

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

  1. Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    SciTech Connect

    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/mg 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.

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

    PubMed

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

    2016-01-01

    Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in 'Valencia' C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of 'Valencia' C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid 'Valencia' C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in 'Valencia' sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in 'Valencia' sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites. PMID:26977938

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

    PubMed

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

    2016-01-01

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

  4. Lymphokine-activated killer (LAK) cells can be focused at sites of tumor growth by products of macrophage activation

    SciTech Connect

    Migliori, R.J.; Gruber, S.A.; Sawyer, M.D.; Hoffman, R.; Ochoa, A.; Bach, F.H.; Simmons, R.L.

    1987-08-01

    Successful adoptive cancer immunotherapy presumably depends on the accumulation of tumoricidal leukocytes at the sites of tumor growth. Large numbers of lymphokine-activated killer (LAK) cells can be generated in vitro by growth in high concentrations of interleukin-2 (IL-2), but relatively few arrive at the tumor site after intravenous injection. We hypothesize that the delivery of LAK cells to tumor sites may be augmented by previously demonstrated lymphocyte-recruiting factors, including activated macrophage products such as interleukin-1 (IL-1) and tumor necrosis factor. /sup 111/Indium-labeled LAK cells were injected intravenously into syngeneic mice bearing the macrophage activator endotoxin (LPS) in one hind footpad, and saline solution was injected into the contralateral footpad. Significantly more activity was recovered from the LPS-bearing footpad at all times during a 96-hour period. Recombinant IL-1 also attracted more LAK cells after injection into tumor-free hind footpads. Furthermore, LAK cells preferentially homed to hind footpads that were bearing 3-day established sarcomas after intralesional injections of LPS, IL-1, or tumor necrosis factor when compared with contralateral tumor-bearing footpads injected with saline solution alone. In preliminary experiments, mice with hind-footpad tumors appeared to survive longer after combined systemic IL-2 and LAK therapy if intralesional LPS was administered. These studies demonstrate that macrophage activation factors that have been shown capable of attracting circulating normal lymphocytes can also effectively attract LAK cells from the circulation. By the stimulation of macrophages at the sites of tumor growth, more LAK cells can be attracted. It is hoped that by focusing the migration of LAK cells to tumors, LAK cells and IL-2 would effect tumor regression more efficiently and with less toxicity.

  5. Solifluction activity in the present periglacial belt of Sierra Nevada during the last 8 ky BP

    NASA Astrophysics Data System (ADS)

    Oliva, M.; Schulte, L.; Gómez Ortiz, A.

    2009-04-01

    Solifluction records in Sierra Nevada (Andalusia, Spain) reveal a succession of environmental changes during the last millennia in this massif mostly related to Holocene climate dynamics. Geomorphological processes in Sierra Nevada react sensitively to small changes in temperature or moisture regimes, showing the proximity of these processes to their climate boundaries and the small climate range necessary to carry environmental changes in the summits of this massif. Solifluction dynamics in Sierra Nevada is influenced by a complex interaction between environmental factors (slope, vegetation cover, texture) and climate parameters (ground thermal regime, length and thickness of the snow cover, water supply). Interdependant feed-back mechanisms among all these variables make difficult to understand the key factors involved in present and past solifluction processes, although monitoring control performed on lobes with different emplacements suggest today's favourable environmental conditions for solifluction displacements. Water availability controls both vegetation cover and slope processes. In fact, currently water supply determines the grass cover in gentle valley floors, but it is also decisive to provide water for the small solifluction displacements detected during the monitored period. Thermal conditions also play a decisive role to activate solifluction or soil formation with similar moisture regimes. The very weak activity pattern of hundreds of solifluction lobes suggests that they must have developed in other more favourable climate conditions. We studied more than 30 sedimentological profiles from solifluction lobes in San Juan and Rio Seco valleys, which reveal an alternation of solifluction/edaphic cycles during the Holocene, with nine different geomorphic phases in the highest western cirques of Sierra Nevada. In San Juan valley, north exposed, there are several generations of solifluction lobes covering the last 8-9 ky BP, while in the southern Rio

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

    PubMed Central

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

    2014-01-01

    The ability to make behavioural inferences from skeletal remains is critical to understanding the lifestyles and activities of past human populations and extinct animals. Muscle attachment site (enthesis) morphology has long been assumed to reflect muscle strength and activity during life, but little experimental evidence exists to directly link activity patterns with muscle development and the morphology of their attachments to the skeleton. We used a mouse model to experimentally test how the level and type of activity influences forelimb muscle architecture of spinodeltoideus, acromiodeltoideus, and superficial pectoralis, bone growth rate and gross morphology of their insertion sites. Over an 11-week period, we collected data on activity levels in one control group and two experimental activity groups (running, climbing) of female wild-type mice. Our results show that both activity type and level increased bone growth rates influenced muscle architecture, including differences in potential muscular excursion (fibre length) and potential force production (physiological cross-sectional area). However, despite significant influences on muscle architecture and bone development, activity had no observable effect on enthesis morphology. These results suggest that the gross morphology of entheses is less reliable than internal bone structure for making inferences about an individual’s past behaviour. PMID:25467113

  7. Satellite imagery time series for the detection of looting activities at archaeological sites

    NASA Astrophysics Data System (ADS)

    Coluzzi, Rosa; Lasaponara, Rosa; Masini, Nicola

    2010-05-01

    Clandestine excavations is one of the biggest man-made risks which affect the archaeological heritage, especially in some countries of Southern America, Asia and Middle East. To contrast and limit this phenomenon a systematic monitoring is required. The protection of archaeological heritage from clandestine excavations is generally based on a direct surveillance, but it is time consuming and expensive for remote archaeological sites and non practicable in several countries due to military or political restrictions. In such conditions, Very high resolution (VHR) satellite imagery offer a suitable chance thanks to their global coverage and frequent revisitation times. This paper is focused on the results we obtained from ongoing research focused on the use of VHR satellite images for the identification and monitoring of looting. A time series of satellite images (QuickBird-2 and World-View-1) has been exploited to analyze and monitor archaeological looting in the Nasca Ceremonial Centre of Cahuachi (Peru) dating back between the 4th centurt B.C. and the 4th century A.D. The Cahuachi study case herein presented put in evidence the limits of VHR satellite imagery in detecting features linked to looting activity. This suggested to experience local spatial autocorrelation statistics which allowed us to improve the reliability of satellite in mapping looted area.

  8. The Role of OOH Binding Site and Pt Surface Structure on ORR Activities

    PubMed Central

    Jia, Qingying; Caldwell, Keegan; Ziegelbauer, Joseph M.; Kongkanand, Anusorn; Wagner, Frederick T.; Mukerjee, Sanjeev; Ramaker, David E.

    2015-01-01

    We present experimentally observed molecular adsorbate coverages (e.g., O(H), OOH and HOOH) on real operating dealloyed bimetallic PtMx (M = Ni or Co) catalysts under oxygen reduction reaction (ORR) conditions obtained using X-ray absorption near edge spectroscopy (XANES). The results reveal a complex Sabatier catalysis behavior and indicate the active ORR mechanism changes with Pt–O bond weakening from the O2 dissociative mechanism, to the peroxyl mechanism, and finally to the hydrogen peroxide mechanism. An important rearrangement of the OOH binding site, an intermediate in the ORR, enables facile H addition to OOH and faster O–O bond breaking on 111 faces at optimal Pt–O bonding strength, such as that occurring in dealloyed PtM core-shell nanoparticles. This rearrangement is identified by previous DFT calculations and confirmed from in situ measured OOH adsorption coverages during the ORR. The importance of surface structural effects and 111 ordered faces is confirmed by the higher specific ORR rates on solid core vs porous multi-core nanoparticles. PMID:26190857

  9. Asymmetry of the active site loop conformation between subunits of glutamate-1-semialdehyde aminomutase in solution.

    PubMed

    Campanini, Barbara; Bettati, Stefano; di Salvo, Martino Luigi; Mozzarelli, Andrea; Contestabile, Roberto

    2013-01-01

    Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5'-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5'-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.

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

    PubMed Central

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

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

  11. Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

    PubMed

    Gross, M; Whetzel, N K; Folk, J E

    1975-10-10

    Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate. PMID:240837

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

    PubMed

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

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

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

  14. Alternative poly(A) site utilization during adenovirus infection coincides with a decrease in the activity of a poly(A) site processing factor.

    PubMed Central

    Mann, K P; Weiss, E A; Nevins, J R

    1993-01-01

    The recognition and processing of a pre-mRNA to create a poly(A) addition site, a necessary step in mRNA biogenesis, can also be a regulatory event in instances in which the frequency of use of a poly(A) site varies. One such case is found during the course of an adenovirus infection. Five poly(A) sites are utilized within the major late transcription unit to produce more than 20 distinct mRNAs during the late phase of infection. The proximal half of the major late transcription unit is also expressed during the early phase of a viral infection. During this early phase of expression, the L1 poly(A) site is used three times more frequently than the L3 poly(A) site. In contrast, the L3 site is used three times more frequently than the L1 site during the late phase of infection. Recent experiments have suggested that the recognition of the poly(A) site GU-rich downstream element by the CF1 processing factor may be a rate-determining step in poly(A) site selection. We demonstrate that the interaction of CF1 with the L1 poly(A) site is less stable than the interaction of CF1 with the L3 poly(A) site. We also find that there is a substantial decrease in the level of CF1 activity when an adenovirus infection proceeds to the late phase. We suggest that this reduction in CF1 activity, coupled with the relative instability of the interaction with the L1 poly(A) site, contributes to the reduced use of the L1 poly(A) site during the late stage of an adenovirus infection. Images PMID:8384308

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

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

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

    PubMed

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

    2016-04-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

  17. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    SciTech Connect

    Parker, Shane M.; Shiozaki, Toru

    2014-12-07

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μE{sub h} or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation.

  18. Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome

    PubMed Central

    Lebailly, Elise; Pages, Carine; Cornet, Francois; Cosentino Lagomarsino, Marco

    2016-01-01

    Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle. PMID:27171414

  19. Functional mimicry of the active site of glutathione peroxidase by glutathione imprinted selenium-containing protein.

    PubMed

    Liu, Lei; Mao, Shi-zhong; Liu, Xiao-man; Huang, Xin; Xu, Jia-yun; Liu, Jun-qiu; Luo, Gui-min; Shen, Jia-cong

    2008-01-01

    For imitating the active site of antioxidant selenoenzyme glutathione peroxidase (GPx), an artificial enzyme selenosubtilisin was employed as a scaffold for reconstructing substrate glutathione (GSH) specific binding sites by a bioimprinting strategy. GSH was first covalently linked to selenosubtilisin to form a covalent complex GSH-selenosubtilisin through a Se-S bond, then the GSH molecule was used as a template to cast a complementary binding site for substrate GSH recognition. The bioimprinting procedure consists of unfolding the conformation of selenosubtilisin and fixing the new conformation of the complex GSH-selenosubtilisin. Thus a new specificity for naturally occurring GPx substrate GSH was obtained. This bioimprinting procedure facilitates the catalytic selenium moiety of the imprinted selenosubtilisin to match the reactive thiol group of GSH in the GSH binding site, which contributes to acceleration of the intramolecular catalysis. These imprinted selenium-containing proteins exhibited remarkable rate enhancement for the reduction of H2O2 by GSH. The average GPx activity was found to be 462 U/micromol, and it was approximately 100 times that for unimprinted selenosubtilisin. Compared with ebselen, a well-known GPx mimic, an activity enhancement of 500-fold was observed. Detailed steady-state kinetic studies demonstrated that the novel selenoenzyme followed a ping-pong mechanism similar to the naturally occurring GPx. PMID:18163571

  20. Evidence for Oxygen Binding at the Active Site of Particulate Methane Monooxygenase

    PubMed Central

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

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. The enzyme consists of three subunits, pmoB, pmoA, and pmoC, organized in an α3β3γ3 trimer. Studies of intact pMMO and a recombinant soluble fragment of the pmoB subunit, denoted spmoB, indicate that the active site is located within the soluble region of pmoB at the site of a crystallographically modeled dicopper center. In this work, we have investigated the reactivity of pMMO and spmoB with oxidants. Upon reduction and treatment of spmoB with O2 and H2O2 or pMMO with H2O2, an absorbance feature at 345 nm is generated. The energy and intensity of this band are similar to that of the μ-η2:η2-peroxo CuII 2 species formed in several dicopper enzymes and model compounds. The feature is not observed in inactive spmoB variants in which the dicopper center is disrupted, consistent with O2 binding to the proposed active site. Reaction of the 345 nm species with CH4 results in disappearance of the spectroscopic feature, suggesting that this O2 intermediate is mechanistically relevant. Taken together, these observations provide strong new support for the identity and location of the pMMO active site. PMID:22540911

  1. Investigating the present and past glacial and frost activity on Pluto with a volatile transport model

    NASA Astrophysics Data System (ADS)

    Bertrand, Tanguy; Forget, Francois

    2016-10-01

    The high obliquity and eccentricity of the orbit of Pluto induce seasonal cycles of condensation and sublimation of the main volatile ices: N2, CH4, and CO. The New Horizons spacecraft, which flew by Pluto in July 2015, revealed a complex surface composition including a thousand-kilometre nitrogen glacier in the "Sputnik Planum" plain near the Anti-Charon longitude, extensive methane frosts at mid and high latitudes, and equatorial ice-free regions. We present numerical simulations designed to model the evolution of Pluto's volatiles over thousands of years on the basis of straightforward universal physical equations.Our results explain the observed distribution of ices on the surface and the quantities of volatiles in the atmosphere. In particular the model predicts the N2 ice accumulation in the deepest low-latitude basin and the 3-fold increase of pressure observed to occur since 1988. This points to atmospheric-topographic processes at the origin of the Sputnik Planum's nitrogen glacier. The same simulations also show frosts of methane, and sometimes nitrogen, that seasonally cover the mid and high latitudes, explaining the bright northern polar cap reported in the 1990s and the observed ice distribution in 2015. The model also predicts that most of these seasonal frosts should disappear in the next decade, and thus could be tested observationally in the near future.Using prior orbital parameters of Pluto and a realistic glacial flow parametrization, we also simulate past climates of Pluto. The results show that Pluto undergoes cycles of glacial activity (over timescales of few million years) that may explain the rugged eroded-mountain landscapes surrounding Sputnik Planum and the "bladed" methane terrains east of "Tombaugh Regio".

  2. Present-Day Seasonal Gully Activity in a South Polar Pit (Sisyphi Cavi) on Mars

    NASA Astrophysics Data System (ADS)

    Raack, Jan; Reiss, Dennis; Appéré, Thomas; Vincendon, Mathieu; Ruesch, Ottaviano; Hiesinger, Harald

    2014-05-01

    Seasonal activity of gullies under current climatic conditions on Mars was observed by [1-7]. Dundas et al. [2] reviewed the present-day activity of classical gullies (including the gully presented in this work), dune gullies, and other mass wasting processes in the southern hemisphere on Mars. Recent polar gullies in Sisyphi Cavi were also analyzed by [8], who estimated ages of about 20 ka to 20 Ma for the gullies. In this study we focus on a single gully in Sisyphi Cavi, located in the south polar region at 1.44° E and 68.54° S. The gully occurs on the gullied equator-facing slope of an isolated polar pit within an infilled impact crater. Multi-temporal high-resolution image data analyses show new deposits at the terminus of the gully channel and on the gully apron within spring (after solar longitudes of 236°) of martian years (MY) 29 and 31. In MY 29 deposition of material shortens the channel by about 40 m; in MY 31 a new deposit at the western flank of the gully apron with approximately 300-600 m3 of material is visible [3]. Our morphological investigations show that the identified new deposits were formed by dark flows through the entire gully deposited on top of the apron between LS ~218° and ~226°. Thermal data show a temperature increase between solar longitudes (LS) ~218° and ~226°. Near-infrared spectral data show relatively constant band strengths of CO2 ice and H2O ice in this time range. After the formation of the dark flows (after LS ~226°), temperatures increase rapidly from ~180 K to >~270 K at LS ~250°. At this time, spectral data indicate that all volatiles on the surface sublimated. However, an earlier beginning of sublimation when the dark flows were observed (between LS ~218° and ~226°) is likely, due to the fact that the instruments can only show the last phase of sublimation (decrease of volatile band strengths) [3]. Spectral modeling shows that from winter to mid-spring, the surface of the studied area is covered by CO2 slab

  3. Activation of human 5-hydroxytryptamine type 3 receptors via an allosteric transmembrane site.

    PubMed

    Lansdell, Stuart J; Sathyaprakash, Chaitra; Doward, Anne; Millar, Neil S

    2015-01-01

    In common with other members of the Cys-loop family of pentameric ligand-gated ion channels, 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are activated by the binding of a neurotransmitter to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. In addition, a variety of compounds have been identified that modulate agonist-evoked responses of 5-HT3Rs, and other Cys-loop receptors, by binding to distinct allosteric sites. In this study, we examined the pharmacological effects of a group of monoterpene compounds on recombinant 5-HT3Rs expressed in Xenopus oocytes. Two phenolic monoterpenes (carvacrol and thymol) display allosteric agonist activity on human homomeric 5-HT3ARs (64 ± 7% and 80 ± 4% of the maximum response evoked by the endogenous orthosteric agonist 5-HT, respectively). In addition, at lower concentrations, where agonist effects are less apparent, carvacrol and thymol act as potentiators of responses evoked by submaximal concentrations of 5-HT. By contrast, carvacrol and thymol have no agonist or potentiating activity on the closely related mouse 5-HT3ARs. Using subunit chimeras containing regions of the human and mouse 5-HT3A subunits, and by use of site-directed mutagenesis, we have identified transmembrane amino acids that either abolish the agonist activity of carvacrol and thymol on human 5-HT3ARs or are able to confer this property on mouse 5-HT3ARs. By contrast, these mutations have no significant effect on orthosteric activation of 5-HT3ARs by 5-HT. We conclude that 5-HT3ARs can be activated by the binding of ligands to an allosteric transmembrane site, a conclusion that is supported by computer docking studies. PMID:25338672

  4. Final Report - Independent Verification Survey Activities at the Seperations Process Research Unit Sites, Niskayuna, New York

    SciTech Connect

    Evan Harpenau

    2011-03-15

    The Separations Process Research Unit (SPRU) complex located on the Knolls Atomic Power Laboratory (KAPL) site in Niskayuna, New York, was constructed in the late 1940s to research the chemical separation of plutonium and uranium (Figure A-1). SPRU operated as a laboratory scale research facility between February 1950 and October 1953. The research activities ceased following the successful development of the reduction oxidation and plutonium/uranium extraction processes. The oxidation and extraction processes were subsequently developed for large scale use by the Hanford and Savannah River sites (aRc 2008a). Decommissioning of the SPRU facilities began in October 1953 and continued through the 1990s.

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

    SciTech Connect

    Petersen, C.A.

    1996-09-20

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

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

    SciTech Connect

    Petersen, C.A., Westinghouse Hanford

    1996-07-17

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

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

    SciTech Connect

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

    2011-08-16

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  10. Function of the active site lysine autoacetylation in Tip60 catalysis.

    PubMed

    Yang, Chao; Wu, Jiang; Zheng, Y George

    2012-01-01

    The 60-kDa HIV-Tat interactive protein (Tip60) is a key member of the MYST family of histone acetyltransferases (HATs) that plays critical roles in multiple cellular processes. We report here that Tip60 undergoes autoacetylation at several lysine residues, including a key lysine residue (i.e. Lys-327) in the active site of the MYST domain. The mutation of K327 to arginine led to loss of both the autoacetylation activity and the cognate HAT activity. Interestingly, deacetylated Tip60 still kept a substantial degree of HAT activity. We also investigated the effect of cysteine 369 and glutamate 403 in Tip60 autoacetylation in order to understand the molecular pathway of the autoacetylation at K327. Together, we conclude that the acetylation of K327 which is located in the active site of Tip60 regulates but is not obligatory for the catalytic activity of Tip60. Since acetylation at this key residue appears to be evolutionarily conserved amongst all MYST proteins, our findings provide an interesting insight into the regulatory mechanism of MYST activities. PMID:22470428

  11. An enzyme activity capable of endotransglycosylation of heteroxylan polysaccharides is present in plant primary cell walls.

    PubMed

    Johnston, Sarah L; Prakash, Roneel; Chen, Nancy J; Kumagai, Monto H; Turano, Helen M; Cooney, Janine M; Atkinson, Ross G; Paull, Robert E; Cheetamun, Roshan; Bacic, Antony; Brummell, David A; Schröder, Roswitha

    2013-01-01

    Heteroxylans in the plant cell wall have been proposed to have a role analogous to that of xyloglucans or heteromannans, forming growth-restraining networks by interlocking cellulose microfibrils. A xylan endotransglycosylase has been identified that can transglycosylate heteroxylan polysaccharides in the presence of xylan-derived oligosaccharides. High activity was detected in ripe fruit of papaya (Carica papaya), but activity was also found in a range of other fruits, imbibed seeds and rapidly growing seedlings of cereals. Xylan endotransglycosylase from ripe papaya fruit used a range of heteroxylans, such as wheat arabinoxylan, birchwood glucuronoxylan and various heteroxylans from dicotyledonous primary cell walls purified from tomato and papaya fruit, as donor molecules. As acceptor molecules, the enzyme preferentially used xylopentaitol over xylohexaitol or shorter-length acceptors. Xylan endotransglycosylase was active over a broad pH range and could perform transglycosylation reactions up to 55 °C. Xylan endotransglycosylase activity was purified from ripe papaya fruit by ultrafiltration and cation exchange chromatography. Highest endotransglycosylase activity was identified in fractions that also contained high xylan hydrolase activity and correlated with the presence of the endoxylanase CpaEXY1. Recombinant CpaEXY1 protein transiently over-expressed in Nicotiana benthamiana leaves showed both endoxylanase and xylan endotransglycosylase activities in vitro, suggesting that CpaEXY1 is a single enzyme with dual activity in planta. Purified native CpaEXY1 showed two- to fourfold higher endoxylanase than endotransglycosylase activity, suggesting that CpaEXY1 may act primarily as a hydrolase. We propose that xylan endotransglycosylase activity (like xyloglucan and mannan endotransglycosylase activities) could be involved in remodelling or re-arrangement of heteroxylans of the cellulose-non-cellulosic cell wall framework.

  12. Purification and characterization of multisquamase, the prothrombin activator present in Echis multisquamatus venom.

    PubMed

    Petrovan, R J; Govers-Riemslag, J W; Nowak, G; Hemker, H C; Rosing, J; Tans, G

    1997-11-01

    The venom of Echis multisquamatus (Central Asian sand viper) contains a single prothrombin activator, designated multisquamase, which is structurally and functionally different from ecarin, the prothrombin activator from the venom of Echis carinatus (saw-scaled viper). Multisquamase is comprised of a 58000 Mr and a 23000 Mr subunit that consists of two disulfide-linked chains of 12000 Mr and 10000 Mr, respectively. In contrast to ecarin, which activates prothrombin and prethrombin 1 at comparable rates, and whose activity is hardly affected by Ca2+ or by changes in ionic strength, multisquamase hardly activates prethrombin 1; prothrombin activation requires Ca2+ and is strongly inhibited at high ionic strength. The most favourable kinetic parameters are observed at 1 mM Ca2+ and at low ionic strength (Km=0.085 microM and kcat=0.68 s(-1) at I approximately 0.04). An increase in ionic strength considerably reduces the rate of prothrombin activation, due to an increase of the Km (Km=0.8 microM and kcat=1.03 s(-1) at I approximately 0.2). Studies in plasmas from patients on oral anticoagulant therapy show that E. Multisquamatus venom only activates carboxylated prothrombin, whereas E. carinatus activates both prothrombin and descarboxyprothrombin. Thus, multisquamase-dependent prothrombin activation appears to require post-translational modification of the gla-domain. This venom prothrombin activator may, therefore, become a useful tool to quantitate prothrombin and descarboxyprothrombin in cases where vitamin K-dependent carboxylation of prothrombin is impaired. PMID:9526951

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

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

  14. The two active sites in human branched-chain alpha-keto acid dehydrogenase operate independently without an obligatory alternating-site mechanism.

    PubMed

    Li, Jun; Machius, Mischa; Chuang, Jacinta L; Wynn, R Max; Chuang, David T

    2007-04-20

    A long standing controversy is whether an alternating activesite mechanism occurs during catalysis in thiamine diphosphate (ThDP)-dependent enzymes. We address this question by investigating the ThDP-dependent decarboxylase/dehydrogenase (E1b) component of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC). Our crystal structure reveals that conformations of the two active sites in the human E1b heterotetramer harboring the reaction intermediate are identical. Acidic residues in the core of the E1b heterotetramer, which align with the proton-wire residues proposed to participate in active-site communication in the related pyruvate dehydrogenase from Bacillus stearothermophilus, are mutated. Enzyme kinetic data show that, except in a few cases because of protein misfolding, these alterations are largely without effect on overall activity of BCKDC, ruling out the requirement of a proton-relay mechanism in E1b. BCKDC overall activity is nullified at 50% phosphorylation of E1b, but it is restored to nearly half of the pre-phosphorylation level after dissociation and reconstitution of BCKDC with the same phosphorylated E1b. The results suggest that the abolition of overall activity likely results from the specific geometry of the half-phosphorylated E1b in the BCKDC assembly and not due to a disruption of the alternating active-site mechanism. Finally, we show that a mutant E1b containing only one functional active site exhibits half of the wild-type BCKDC activity, which directly argues against the obligatory communication between active sites. The above results provide evidence that the two active sites in the E1b heterotetramer operate independently during the ThDP-dependent decarboxylation reaction. PMID:17329260

  15. Linde FUSRAP Site Remediation: Engineering Challenges and Solutions of Remedial Activities on an Active Industrial Facility - 13506

    SciTech Connect

    Beres, Christopher M.; Fort, E. Joseph; Boyle, James D.

    2013-07-01

    The Linde FUSRAP Site (Linde) is located in Tonawanda, New York at a major research and development facility for Praxair, Inc. (Praxair). Successful remediation activities at Linde combines meeting cleanup objectives of radiological contamination while minimizing impacts to Praxair business operations. The unique use of Praxair's property coupled with an array of active and abandoned utilities poses many engineering and operational challenges; each of which has been overcome during the remedial action at Linde. The U.S. Army Corps of Engineers - Buffalo District (USACE) and CABRERA SERVICES, INC. (CABRERA) have successfully faced engineering challenges such as relocation of an aboveground structure, structural protection of an active water line, and installation of active mechanical, electrical, and communication utilities to perform remediation. As remediation nears completion, continued success of engineering challenges is critical as remaining activities exist in the vicinity of infrastructure essential to business operations; an electrical substation and duct bank providing power throughout the Praxair facility. Emphasis on engineering and operations through final remediation and into site restoration will allow for the safe and successful completion of the project. (authors)

  16. Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

    SciTech Connect

    Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.; Elsen, Nathaniel L.; Phillips, Jr., George N.; Fox, Brian G.

    2014-10-02

    Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric {mu}-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH{sub 2}-benzoate and p-Br-benzoate showed a {mu}-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a {pi}-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 {angstrom}) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential

  17. Active Site Dependent Reaction Mechanism over Ru/CeO2 Catalyst toward CO2 Methanation.

    PubMed

    Wang, Fei; He, Shan; Chen, Hao; Wang, Bin; Zheng, Lirong; Wei, Min; Evans, David G; Duan, Xue

    2016-05-18

    Oxygen vacancy on the surface of metal oxides is one of the most important defects which acts as the reactive site in a variety of catalytic reactions. In this work, operando spectroscopy methodology was employed to study the CO2 methanation reaction catalyzed by Ru/CeO2 (with oxygen vacancy in CeO2) and Ru/α-Al2O3 (without oxygen vacancy), respectively, so as to give a thorough understanding on active site dependent reaction mechanism. In Ru/CeO2 catalyst, operando XANES, IR, and Raman were used to reveal the generation process of Ce(3+), surface hydroxyl, and oxygen vacancy as well as their structural evolvements under practical reaction conditions. The steady-state isotope transient kinetic analysis (SSITKA)-type in situ DRIFT infrared spectroscopy undoubtedly substantiates that CO2 methanation undergoes formate route over Ru/CeO2 catalyst, and the formate dissociation to methanol catalyzed by oxygen vacancy is the rate-determining step. In contrast, CO2 methanation undergoes CO route over Ru surface in Ru/α-Al2O3 with the absence of oxygen vacancy, demonstrating active site dependent catalytic mechanism toward CO2 methanation. In addition, the catalytic activity evaluation and the oscillating reaction over Ru/CeO2 catalyst further prove that the oxygen vacancy catalyzes the rate-determining step with a much lower activation temperature compared with Ru surface in Ru/α-Al2O3 (125 vs 250 °C).

  18. Progress report on decommissioning activities at the Fernald Environmental Management Project (FEMP) site

    SciTech Connect

    1998-07-01

    The Fernald Environmental Management Project (FEMP), is located about 18 miles northwest of Cincinnati, Ohio. Between 1953 and 1989, the facility, then called the Feed Material Production Center or FMPC, produced uranium metal products used in the eventual production of weapons grade material for use by other US Department of Energy (DOE) sites. In 1989, FMPC`s production was suspended by the federal government in order to focus resources on environmental restoration versus defense production. In 1992, Fluor Daniel Fernald assumed responsibility for managing all cleanup activities at the FEMP under contract to the DOE. In 1990, as part of the remediation effort, the site was divided into five operable units based on physical proximity of contaminated areas, similar amounts of types of contamination, or the potential for a similar technology to be used in cleanup activities. This report continues the outline of the decontamination and decommissioning (D and D) activities at the FEMP site Operable Unit 3 (OU3) and provides an update on the status of the decommissioning activities. OU3, the Facilities Closure and Demolition Project, involves the remediation of more than 200 uranium processing facilities. The mission of the project is to remove nuclear materials stored in these buildings, then perform the clean out of the buildings and equipment, and decontaminate and dismantle the facilities.

  19. Identification of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase.

    PubMed

    Batt, C A; Jamieson, A C; Vandeyar, M A

    1990-01-01

    Two conserved histidine residues (His-101 and His-271) appear to be essential components in the active site of the enzyme xylose (glucose) isomerase (EC 5.3.1.5). These amino acid residues were targeted for mutagenesis on the basis of sequence homology among xylose isomerases isolated from Escherichia coli, Bacillus subtilis, Ampullariella sp. strain 3876, and Streptomyces violaceus-niger. Each residue was selectively replaced by site-directed mutagenesis and shown to be essential for activity. No measurable activity was observed for any mutations replacing either His-101 or His-271. Circular dichroism measurements revealed no significant change in the overall conformation of the mutant enzymes, and all formed dimers similar to the wild-type enzyme. Mutations at His-271 could be distinguished from those at His-101, since the former resulted in a thermolabile protein whereas no significant change in heat stability was observed for the latter. Based upon these results and structural data recently reported, we speculate that His-101 is the catalytic base mediating the reaction. Replacement of His-271 may render the enzyme thermolabile, since this residue appears to be a ligand for one of the metal ions in the active site of the enzyme. PMID:2405386

  20. Structural basis for the active site inhibition mechanism of human kidney-type glutaminase (KGA).

    PubMed

    Thangavelu, K; Chong, Qing Yun; Low, Boon Chuan; Sivaraman, J

    2014-01-01

    Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the glutaminase isoforms, human kidney-type glutaminase (KGA) is often upregulated in cancer and is thus touted as an attractive drug target. Here we report the active site inhibition mechanism of KGA through the crystal structure of the catalytic domain of KGA (cKGA) in complex with 6-diazo-5-oxo-L-norleucine (DON), a substrate analogue of glutamine. DON covalently binds with the active site Ser286 and interacts with residues such as Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484. The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N2) from the inhibitor and results in the formation of an enzyme-inhibitor complex. Mutational studies confirmed the key role of these residues in the activity of KGA. This study will be important in the development of KGA active site inhibitors for therapeutic interventions.

  1. Structural Basis for the Active Site Inhibition Mechanism of Human Kidney-Type Glutaminase (KGA)

    PubMed Central

    Thangavelu, K.; Chong, Qing Yun; Low, Boon Chuan; Sivaraman, J.

    2014-01-01

    Glutaminase is a metabolic enzyme responsible for glutaminolysis, a process harnessed by cancer cells to feed their accelerated growth and proliferation. Among the glutaminase isoforms, human kidney-type glutaminase (KGA) is often upregulated in cancer and is thus touted as an attractive drug target. Here we report the active site inhibition mechanism of KGA through the crystal structure of the catalytic domain of KGA (cKGA) in complex with 6-diazo-5-oxo-L-norleucine (DON), a substrate analogue of glutamine. DON covalently binds with the active site Ser286 and interacts with residues such as Tyr249, Asn335, Glu381, Asn388, Tyr414, Tyr466 and Val484. The nucleophilic attack of Ser286 sidechain on DON releases the diazo group (N2) from the inhibitor and results in the formation of an enzyme-inhibitor complex. Mutational studies confirmed the key role of these residues in the activity of KGA. This study will be important in the development of KGA active site inhibitors for therapeutic interventions. PMID:24451979

  2. How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism

    PubMed Central

    Hytönen, Vesa P; Vogel, Viola

    2008-01-01

    Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds. PMID:18282082

  3. From single crystal surfaces to single atoms: investigating active sites in electrocatalysis.

    PubMed

    O'Mullane, Anthony P

    2014-04-21

    Electrocatalytic processes will undoubtedly be at the heart of energising future transportation and technology with the added importance of being able to create the necessary fuels required to do so in an environmentally friendly and cost effective manner. For this to be successful two almost mutually exclusive surface properties need to be reconciled, namely producing highly active/reactive surface sites that exhibit long term stability. This article reviews the various approaches which have been undertaken to study the elusive nature of these active sites on metal surfaces which are considered as adatoms or clusters of adatoms with low coordination number. This includes the pioneering studies at extended well defined stepped single crystal surfaces using cyclic voltammetry up to the highly sophisticated in situ electrochemical imaging techniques used to study chemically synthesised nanomaterials. By combining the information attained from single crystal surfaces, individual nanoparticles of defined size and shape, density functional theory calculations and new concepts such as mesoporous multimetallic thin films and single atom electrocatalysts new insights into the design and fabrication of materials with highly active but stable active sites can be achieved. The area of electrocatalysis is therefore not only a fascinating and exciting field in terms of realistic technological and economical benefits but also from the fundamental understanding that can be acquired by studying such an array of interesting materials. PMID:24599277

  4. Novel active comb-shaped dry electrode for EEG measurement in hairy site.

    PubMed

    Huang, Yan-Jun; Wu, Chung-Yu; Wong, Alice May-Kuen; Lin, Bor-Shyh

    2015-01-01

    Electroencephalography (EEG) is an important biopotential, and has been widely applied in clinical applications. The conventional EEG electrode with conductive gels is usually used for measuring EEG. However, the use of conductive gel also encounters with the issue of drying and hardening. Recently, many dry EEG electrodes based on different conductive materials and techniques were proposed to solve the previous issue. However, measuring EEG in the hairy site is still a difficult challenge. In this study, a novel active comb-shaped dry electrode was proposed to measure EEG in hairy site. Different form other comb-shaped or spike-shaped dry electrodes, it can provide more excellent performance of avoiding the signal attenuation, phase distortion, and the reduction of common mode rejection ratio. Even under walking motion, it can effectively acquire EEG in hairy site. Finally, the experiments for alpha rhythm and steady-state visually evoked potential were also tested to validate the proposed electrode.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements of the amount of lipase immobilized (bicinchoninic acid method) and the catalyst’s tributyrin hydrolysis activity, coupled with a determination of the lipase’s functional fr...

  6. Pattern formation for active particles on optically created ordered and disordered substrates (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    Reichhardt, Charles M.; Ray, Dipanjan; Reichhardt, Cynthia J.

    2015-08-01

    There has been tremendous growth in the field of active matter, where the individual particles that comprise the system are self-driven. Examples of this class of system include biological systems such as swimming bacteria and crawling cells. More recently, non-biological swimmers have been created using colloidal Janus particles that undergo chemical reactions on one side to produce self-propulsion. These active matter systems exhibit a wide variety of behaviors that are absent in systems undergoing purely thermal fluctuations, such as transitions from uniform liquids to clusters or living crystals, pushing objects around, ratchet effects, and phase separation in mixtures of active and passive particles. Here we examine the collective effects of active matter disks in the presence of static or dynamic substrates. For colloids, such substrates could be created optically in the form of periodic, random, or quasiperiodic patterns. For thermal particles, increasing the temperature generally increases the diffusion or mobility of the particles when they move over a random or periodic substrates. We find that when the particles are active, increasing the activity can increase the mobility for smaller run lengths but decrease the mobility at large run lengths. Additionally we find that at large run lengths on a structured substrate, a variety of novel active crystalline states can form such as stripes, squares and triangular patterns.

  7. Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-(p-(fluorosulfonyl)benzoyl)adenosine

    SciTech Connect

    Phelps, D.C.; Hatefi, Y.

    1985-07-02

    Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-(p-(fluorosulfonyl)benzoyl)adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. (/sup 3/H)FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of (3H)FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by (/sup 14/C)DCCD. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.

  8. Evidence for large earthquakes on the San Andreas fault at the Wrightwood, California paleoseismic site: A.D. 500 to present

    USGS Publications Warehouse

    Fumal, T.E.; Weldon, R.J.; Biasi, G.P.; Dawson, T.E.; Seitz, G.G.; Frost, W.T.; Schwartz, D.P.

    2002-01-01

    We present structural and stratigraphic evidence from a paleoseismic site near Wrightwood, California, for 14 large earthquakes that occurred on the southern San Andreas fault during the past 1500 years. In a network of 38 trenches and creek-bank exposures, we have exposed a composite section of interbedded debris flow deposits and thin peat layers more than 24 m thick; fluvial deposits occur along the northern margin of the site. The site is a 150-m-wide zone of deformation bounded on the surface by a main fault zone along the northwest margin and a secondary fault zone to the southwest. Evidence for most of the 14 earthquakes occurs along structures within both zones. We identify paleoearthquake horizons using infilled fissures, scarps, multiple rupture terminations, and widespread folding and tilting of beds. Ages of stratigraphic units and earthquakes are constrained by historic data and 72 14C ages, mostly from samples of peat and some from plant fibers, wood, pine cones, and charcoal. Comparison of the long, well-resolved paleoseimic record at Wrightwood with records at other sites along the fault indicates that rupture lengths of past earthquakes were at least 100 km long. Paleoseismic records at sites in the Coachella Valley suggest that each of the past five large earthquakes recorded there ruptured the fault at least as far northwest as Wrightwood. Comparisons with event chronologies at Pallett Creek and sites to the northwest suggests that approximately the same part of the fault that ruptured in 1857 may also have failed in the early to mid-sixteenth century and several other times during the past 1200 years. Records at Pallett Creek and Pitman Canyon suggest that, in addition to the 14 earthquakes we document, one and possibly two other large earthquakes ruptured the part of the fault including Wrightwood since about A.D. 500. These observations and elapsed times that are significantly longer than mean recurrence intervals at Wrightwood and sites to

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

    PubMed Central

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

    2000-01-01

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

  10. 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.

  11. Immobilization of alkaline phosphatase on solid surface through self-assembled monolayer and by active-site protection.

    PubMed

    Gao, En-Feng; Kang, Kyung Lhi; Kim, Jeong Hee

    2014-06-01

    Retaining biological activity of a protein after immobilization is an important issue and many studies reported to enhance the activity of proteins after immobilization. We recently developed a new immobilization method of enzyme using active-site protection and minimization of the cross-links between enzyme and surface with a DNA polymerase as a model system. In this study, we extended the new method to an enzyme with a small mono-substrate using alkaline phosphatase (AP) as another model system. A condition to apply the new method is that masking agents, in this case its own substrate needs to stay at the active-site of the enzyme to be immobilized in order to protect the active-site during the harsh immobilization process. This could be achieved by removal of essential divalent ion, Zn2+ that is required for full enzyme activity of AP from the masking solution while active-site of AP was protected with p-nitrophenyl phosphate (pNPP). Approximately 40% of the solution-phase activity was acquired with active-site protected immobilized AP. In addition to protection active-site of AP, the number of immobilization links was kinetically controlled. When the mole fraction of the activated carboxyl group of the linker molecule in self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 6-mercapto-1-ethanol was varied, 10% of 12-mercaptododecanoic acid gave the maximum enzyme activity. Approximately 51% increase in enzyme activity of the active-site protected AP was observed compared to that of the unprotected group. It was shown that the concept of active-site protection and kinetic control of the number of covalent immobilization bonds can be extended to enzymes with small mono-substrates. It opens the possibility of further extension of the new methods of active-site protection and kinetic control of immobilization bond to important enzymes used in research and industrial fields. PMID:24738440

  12. Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

    PubMed Central

    Vallejos, Maricarmen; Carvajal, Felipe; Pino, Karla; Navarrete, Camilo; Ferres, Marcela; Huidobro-Toro, Juan Pablo; Sargueil, Bruno; López-Lastra, Marcelo

    2012-01-01

    The 5′untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5′UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to crypti