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Sample records for active sites responsible

  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. Failure of origin activation in response to fork stalling leads to chromosomal instability at fragile sites.

    PubMed

    Ozeri-Galai, Efrat; Lebofsky, Ronald; Rahat, Ayelet; Bester, Assaf C; Bensimon, Aaron; Kerem, Batsheva

    2011-07-01

    Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Here, we show that even under normal growth conditions, replication fork progression along the fragile site, FRA16C, is slow and forks frequently stall at AT-rich sequences, leading to activation of additional origins to enable replication completion. Under mild replication stress, the frequency of stalling at AT-rich sequences is further increased. Strikingly, unlike in the entire genome, in the FRA16C region additional origins are not activated, suggesting that all potential origins are already activated under normal conditions. Thus, the basis for FRA16C fragility is replication fork stalling at AT-rich sequences and inability to activate additional origins under replication stress. Our results provide a mechanism explaining the replication stress sensitivity of fragile sites and thus, the basis for genomic instability during early stages of cancer development. PMID:21726815

  3. Protein oxidation mediated by heme-induced active site conversion specific for heme-regulated transcription factor, iron response regulator

    PubMed Central

    Kitatsuji, Chihiro; Izumi, Kozue; Nambu, Shusuke; Kurogochi, Masaki; Uchida, Takeshi; Nishimura, Shin-Ichiro; Iwai, Kazuhiro; O’Brian, Mark R.; Ikeda-Saito, Masao; Ishimori, Koichiro

    2016-01-01

    The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O2 to form highly reactive oxygen species (ROS) with the “active site conversion” from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H2O2 from O2 as found for other hemoproteins, but H2O2 is less effective in oxidizing the peptide, and further activation of H2O2 is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel “two-step self-oxidative modification” mechanism, during which O2 is activated to form H2O2 at the heme regulatory motif (HRM) site and the generated H2O2 is further converted into more reactive species such as ·OH at the non-heme iron site in the His-cluster region formed by the active site conversion. PMID:26729068

  4. WWOX, the common fragile site FRA16D gene product, regulates ATM activation and the DNA damage response

    PubMed Central

    Abu-Odeh, Mohammad; Salah, Zaidoun; Herbel, Christoph; Hofmann, Thomas G.; Aqeilan, Rami I.

    2014-01-01

    Genomic instability is a hallmark of cancer. The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor spanning the common chromosomal fragile site FRA16D. Here, we report a direct role of WWOX in DNA damage response (DDR) and DNA repair. We show that Wwox deficiency results in reduced activation of the ataxia telangiectasia-mutated (ATM) checkpoint kinase, inefficient induction and maintenance of γ-H2AX foci, and impaired DNA repair. Mechanistically, we show that, upon DNA damage, WWOX accumulates in the cell nucleus, where it interacts with ATM and enhances its activation. Nuclear accumulation of WWOX is regulated by its K63-linked ubiquitination at lysine residue 274, which is mediated by the E3 ubiquitin ligase ITCH. These findings identify a novel role for the tumor suppressor WWOX and show that loss of WWOX expression may drive genomic instability and provide an advantage for clonal expansion of neoplastic cells. PMID:25331887

  5. Can site response be predicted?

    USGS Publications Warehouse

    Boore, D.M.

    2004-01-01

    Large modifications of seismic waves are produced by variations of material properties near the Earth's surface and by both surface and buried topography. These modifications, usually referred to as "site response", in general lead to larger motions on soil sites than on rock-like sites. Because the soil amplifications can be as large as a factor of ten, they are important in engineering applications that require the quantitative specification of ground motions. This has been recognised for years by both seismologists and engineers, and it is hard to open an earthquake journal these days without finding an article on site response. What is often missing in these studies, however, are discussions of the uncertainty of the predicted response. A number of purely observational studies demonstrate that ground motions have large site-to-site variability for a single earthquake and large earthquake-location- dependent variability for a single site. This variability makes site-specific, earthquake-specific predictions of site response quite uncertain, even if detailed geotechnical and geological information is available near the site. Predictions of site response for average classes of sites exposed to the motions from many earthquakes can be made with much greater certainty if sufficient empirical observations are available.

  6. TOPOGRAPHIC SITE RESPONSE AT HARD ROCK SITES

    NASA Astrophysics Data System (ADS)

    Yong, A. K.; Hough, S. E.

    2009-12-01

    Site (material impedance) and topographic (geometric form) effects are known to be key factors that influence seismic ground motions. To characterize site effects, Yong et al. (2009) developed a terrain-based Vs30 prediction model using an automated classification method (Iwahashi and Pike, 2007) that relied on taxonomic criteria (slope gradient, local convexity and surface texture) developed from geomorphometry to identify 16 terrain types from a 1-km spatial resolution (SRTM30 data) digital elevation model of California. On the basis that the underlying framework for this model contains parameters (esp., local convexity) which aptly describe the geometry (i.e., base to height ratio) of relief features that are known to also control the behavior of ground motions (Bouchon, 1973), we extend our investigation to study topographic effects. Focusing on sites that would generally be considered “hard rock,” the classification scheme distinguishes 7 separate terrain types ranging from “moderately eroded mountains” to “well dissected alpine summits.” Observed 1-Hz amplification factors at Southern California Seismographic Network sites reveal a weak but systematic correlation with these 7 terrain types. Significant scatter is also found within each terrain type; typical standard deviations of logarithmic amplification factors are 0.2-0.3. Considering stations that have high amplification factors, we find some that have apparently been misclassified due to data resolution limitations. Many of the remaining stations with higher than expected amplifications are located on or near topographic peaks or ridges. The unusually high amplification factors at hard-rock sites, typically factors of 1.5-2, can most plausibly be explained as a topographic effect.

  7. Antigenic sites on the HN domain of botulinum neurotoxin A stimulate protective antibody responses against active toxin

    PubMed Central

    Vijayalakshmi Ayyar, B.; Tajhya, Rajeev B.; Beeton, Christine; Zouhair Atassi, M.

    2015-01-01

    Botulinum neurotoxins (BoNTs) are the most toxic substances known. BoNT intoxicates cells in a highly programmed fashion initiated by binding to the cell surface, internalization and enzymatic cleavage of substrate, thus, inhibiting synaptic exocytosis. Over the past two decades, immunological significance of BoNT/A C-terminal heavy chain (HC) and light chain (LC) domains were investigated extensively leading to important findings. In the current work, we explored the significance of BoNT/A heavy chain N-terminal (HN) region as a vaccine candidate. Mice were immunized with recombinant HN519–845 generating antibodies (Abs) that were found to be protective against lethal dose of BoNT/A. Immuno-dominant regions of HN519–845 were identified and individually investigated for antibody response along with synthetic peptides within those regions, using in vivo protection assays against BoNT/A. Results were confirmed by patch-clamp analysis where anti-HN antibodies were studied for the ability to block toxin-induced channel formation. This data strongly indicated that HN519–593 is an important region in generating protective antibodies and should be valuable in a vaccine design. These results are the first to describe and dissect the protective activity of the BoNT/A HN domain. PMID:26508475

  8. Performance of remedial response, activities at uncontrolled hazardous waste sites (REM II) remedial investigation first operable unit, Rocky Mountain Arsenal Offpost R1/fS site

    SciTech Connect

    Smith, M.J.

    1986-12-01

    The objective of this remedial investigation (RI) is to evaluate, as part of the first operable unit for site remediation, the nature and extent of contamination as it relates to the existing public water supply wells operated by the South Adams County Water and Sanitation District. A public health endangerment assessment was conducted to identify compounds which could pose a significant health threat. These investigations have identified ground water as the major migration pathway. The six volatile organic chemicals of primary concern are 1,1-DCE, 1,1-DCLE, t-1,2-DCE, 1,1,1-TCE, TRClE, and TClEE. A comprehensive investigation analysis was conducted to (1) characterize the hydrogeologic system and (2) quantify the ground water flow and containment transport processes. A three dimensional ground water flow and containment transfer model was developed and calibrated on a preliminary basis to aid in projection of future TRClE concentrations. This first operable unit RI report is not in intended to address the identity of contributing sources or their relative contributions to the contamination.

  9. Salt site performance assessment activities

    SciTech Connect

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

    1983-01-01

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

  10. Vs30 and spectral response from collocated shallow, active- and passive-source Vs data at 27 sites in Puerto Rico

    USGS Publications Warehouse

    Odum, Jack K.; Stephenson, William J.; Williams, Robert A.; von Hillebrandt-Andrade, Christa

    2013-01-01

    Shear‐wave velocity (VS) and time‐averaged shear‐wave velocity to 30 m depth (VS30) are the key parameters used in seismic site response modeling and earthquake engineering design. Where VS data are limited, available data are often used to develop and refine map‐based proxy models of VS30 for predicting ground‐motion intensities. In this paper, we present shallow VS data from 27 sites in Puerto Rico. These data were acquired using a multimethod acquisition approach consisting of noninvasive, collocated, active‐source body‐wave (refraction/reflection), active‐source surface wave at nine sites, and passive‐source surface‐wave refraction microtremor (ReMi) techniques. VS‐versus‐depth models are constructed and used to calculate spectral response plots for each site. Factors affecting method reliability are analyzed with respect to site‐specific differences in bedrock VS and spectral response. At many but not all sites, body‐ and surface‐wave methods generally determine similar depths to bedrock, and it is the difference in bedrock VS that influences site amplification. The predicted resonant frequencies for the majority of the sites are observed to be within a relatively narrow bandwidth of 1–3.5 Hz. For a first‐order comparison of peak frequency position, predictive spectral response plots from eight sites are plotted along with seismograph instrument spectra derived from the time series of the 16 May 2010 Puerto Rico earthquake. We show how a multimethod acquisition approach using collocated arrays compliments and corroborates VS results, thus adding confidence that reliable site characterization information has been obtained.

  11. REMEDIAL RESPONSE AT THE RICHMOND, CALIFORNIA HAZARDOUS WASTE SITE

    EPA Science Inventory

    The U.S. Environmental Protection Agency surveyed 395 uncontrolled hazardous waste sites where some form of remedial action had been planned. A series of reports on 23 sites representative of various remedial response activities has been prepared. The current report from this ser...

  12. A dioxin response element in the multiple cloning site of the pGL3 luciferase reporter influences transcriptional activity1

    PubMed Central

    Ochs, Sharon; Liu, Jing; Fernando, Tharu; Fecher, Roger; Sulentic, Courtney E.W.

    2012-01-01

    Luciferase reporter plasmids (pGL3 backbone, Promega) have been utilized to characterize the transcriptional effects of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands. Following ligand activation, the AhR and its dimerization partner AhR nuclear translocator (ARNT) regulate transcription by binding dioxin response elements (DREs) in regulatory regions of dioxin-sensitive genes. Upon sequencing of our luciferase reporters, we unexpectedly identified a DRE core motif within the multiple cloning site (mcsDRE) of the pGL3 luciferase plasmid backbone in a subset of our reporters. Therefore, the objective of this study was to determine if the mcsDRE inadvertently influences reporter activity. Utilizing deletional analysis we determined that the mcsDRE did significantly alter the transcriptional effect induced by TCDD. Since many chemicals have been shown to interact with the AhR and influence transcription through the DRE, the presence of the mcsDRE in the pGL3 luciferase plasmid may inappropriately influence promoter and enhancer analysis. As such, insertion of regulatory elements into pGL3 reporters should be designed to avoid retaining the mcsDRE core motif (GCGTG) and currently utilized pGL3 reporters should be evaluated for the presence of the mcsDRE. PMID:22652426

  13. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

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

    1990-10-01

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

  14. 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.

  15. Serum response element-like sequences of the human low density lipoprotein receptor promoter: possible regulation sites for sterol-independent transcriptional activation.

    PubMed

    Pak, Y K

    1996-02-01

    Serum factors stimulate low density lipoprotein receptor (LDLR) gene expression in HepG2 cells through sterol-independent pathways. Promoter element other than sterol regulatory element-1 (SRE-1) seems to be necessary. Protein binding activity of the human LDLR promoter fragment (550bp) beyond the SRE-1 was determined by DNase I footprint assay. Five different promoter regions were protected from DNase I digestion; -226 to -258, -291 to -304, -324 to -336, -360 to -373, and -521 to -528. The regions of -324 to -336 and -521 to -528 showed serum response element (SRE)-like consensus sequence of CC(A/T)6GG. Serum incubation affected the protection degree of the SRE-like elements, but 25-hydroxycholesterol did not. It is proposed, therefore, that the promoter region of -324 to -336 and/or -521 to -528 showed serum response elements, but 25-hydroxycholesterol did not. It is proposed, therefore, that the promoter region of -324 to -336 and/or -521 to -528 in human LDLR gene may be responsible for the rapid activation of the gene transcription by serum factor in a sterol-independent manner. PMID:8932516

  16. Measures of site resourcing predict virologic suppression, immunologic response and HIV disease progression following highly active antiretroviral therapy (HAART) in the TREAT Asia HIV Observational Database (TAHOD)

    PubMed Central

    Oyomopito, R; Lee, MP; Phanuphak, P; Lim, PL; Ditangco, R; Zhou, J; Sirisanthana, T; Chen, YMA; Pujari, S; Kumarasamy, N; Sungkanuparph, S; Lee, CKC; Kamarulzaman, A; Oka, S; Zhang, FJ; Mean, CV; Merati, T; Tau, G; Smith, J; Li, PCK

    2010-01-01

    Objectives Surrogate markers of HIV disease progression are HIV RNA in plasma viral load (VL) and CD4 cell count (immune function). Despite improved international access to antiretrovirals, surrogate marker diagnostics are not routinely available in resource-limited settings. Therefore, the objective was to assess effects of economic and diagnostic resourcing on patient treatment outcomes. Methods Analyses were based on 2333 patients initiating highly active antiretroviral therapy (HAART) from 2000 onwards. Sites were categorized by World Bank country income criteria (high/low) and annual frequency of VL (≥ 3, 1–2 or <1) or CD4 (≥ 3 or <3) testing. Endpoints were time to AIDS/death and change in CD4 cell count and VL suppression (<400 HIV-1 RNA copies/mL) at 12 months. Demographics, Centers for Disease Control and Prevention (CDC) classification, baseline VL/CD4 cell counts, hepatitis B/C coinfections and HAART regimen were covariates. Time to AIDS/death was analysed by proportional hazards models. CD4 and VL endpoints were analysed using linear and logistic regression, respectively. Results Increased disease progression was associated with site-reported VL testing less than once per year [hazard ratio (HR)=1.4; P=0.032], severely symptomatic HIV infection (HR=1.4; P=0.003) and hepatitis C virus coinfection (HR=1.8; P=0.011). A total of 1120 patients (48.2%) had change in CD4 cell count data. Smaller increases were associated with older age (P<0.001) and `Other' HIV source exposures, including injecting drug use and blood products (P=0.043). A total of 785 patients (33.7%) contributed to the VL suppression analyses. Patients from sites with VL testing less than once per year [odds ratio (OR)=0.30; P<0.001] and reporting `Other' HIV exposures experienced reduced suppression (OR=0.28; P<0.001). Conclusion Low measures of site resourcing were associated with less favourable patient outcomes, including a 35% increase in disease progression in patients from sites

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

    PubMed

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

    2011-01-01

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

  18. Meeting Reference Responsibilities through Library Web Sites.

    ERIC Educational Resources Information Center

    Adams, Michael

    2001-01-01

    Discusses library Web sites and explains some of the benefits when libraries make their sites into reference portals, linking them to other useful Web sites. Topics include print versus Web information sources; limitations of search engines; what Web sites to include, including criteria for inclusions; and organizing the sites. (LRW)

  19. Educational Activity Sites for High School Students

    ERIC Educational Resources Information Center

    Troutner, Joanne

    2005-01-01

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

  20. Development of site-specific earthquake response spectra for eastern US sites

    SciTech Connect

    Beavers, J.E.; Brock, W.R.; Hunt, R.J.; Shaffer, K.E.

    1993-08-01

    Site-specific earthquake, uniform-hazard response spectra have been defined for the Department of Energy Oak Ridge, Tennessee, and Portsmouth, Ohio, sites for use in evaluating existing facilities and designing new facilities. The site-specific response spectra were defined from probabilistic and deterministic seismic hazard studies following the requirements in DOE-STD-1024-92, ``Guidelines for Probabilistic Seismic Hazard Curves at DOE Sites.` For these two sites, the results show that site-specific uniform-hazard response spectra are slightly higher in the high-frequency range and considerably lower in the low-frequency range compared with response spectra defined for these sites in the past.

  1. Site-Specific Seismic Site Response Model for the Waste Treatment Plant, Hanford, Washington

    SciTech Connect

    Rohay, Alan C.; Reidel, Steve P.

    2005-02-24

    This interim report documents the collection of site-specific geologic and geophysical data characterizing the Waste Treatment Plant site and the modeling of the site-specific structure response to earthquake ground motions.

  2. 40 CFR 228.3 - Disposal site management responsibilities.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Disposal site management responsibilities. 228.3 Section 228.3 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) OCEAN DUMPING CRITERIA FOR THE MANAGEMENT OF DISPOSAL SITES FOR OCEAN DUMPING § 228.3 Disposal site management responsibilities. (a) Management of a...

  3. 40 CFR 228.3 - Disposal site management responsibilities.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 25 2014-07-01 2014-07-01 false Disposal site management responsibilities. 228.3 Section 228.3 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) OCEAN DUMPING CRITERIA FOR THE MANAGEMENT OF DISPOSAL SITES FOR OCEAN DUMPING § 228.3 Disposal site management responsibilities. (a) Management of a...

  4. 40 CFR 228.3 - Disposal site management responsibilities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 26 2013-07-01 2013-07-01 false Disposal site management... DUMPING CRITERIA FOR THE MANAGEMENT OF DISPOSAL SITES FOR OCEAN DUMPING § 228.3 Disposal site management responsibilities. (a) Management of a site consists of regulating times, rates, and methods of disposal...

  5. 40 CFR 228.3 - Disposal site management responsibilities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 25 2011-07-01 2011-07-01 false Disposal site management... DUMPING CRITERIA FOR THE MANAGEMENT OF DISPOSAL SITES FOR OCEAN DUMPING § 228.3 Disposal site management responsibilities. (a) Management of a site consists of regulating times, rates, and methods of disposal...

  6. 40 CFR 228.3 - Disposal site management responsibilities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Disposal site management... DUMPING CRITERIA FOR THE MANAGEMENT OF DISPOSAL SITES FOR OCEAN DUMPING § 228.3 Disposal site management responsibilities. (a) Management of a site consists of regulating times, rates, and methods of disposal...

  7. 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

  8. Activation response of martensitic steels

    SciTech Connect

    Forty, C.B.A.

    1997-09-01

    A hypothetical martensitic steel has been compositionally designed in order to optimize both metallurgical and reduced activation properties. When compared with two other martensitic steels, its activation characteristics are shown to be superior for all activation indices examined. However, these excellent properties are found to be due to the assumed absence of deleterious tramp impurities. When limiting impurity concentrations are determined for the hypothetical steel, they are found to be extremely stringent, and wholly unachievable using industrial scale production methods. It is concluded that only slight improvements can be made to currently available low activation martensitic steels to reduce residual activity responses further. 26 refs., 1 fig., 2 tabs.

  9. Activation Response of Martensitic Steels

    NASA Astrophysics Data System (ADS)

    Forty, C. B. A.

    1997-09-01

    A hypothetical martensitic steel has been compositionally designed in order to optimize both metallurgical and reduced activation properties. When compared with two other martensitic steels, its activation characteristics are shown to be superior for all activation indices examined. However, these excellent properties are found to be due to the assumed absence of deleterious tramp impurities. When limiting impurity concentrations are determined for the hypothetical steel, they are found to be extremely stringent, and wholly unachievable using industrial scale production methods. It is concluded that only slight improvements can be made to currently available low activation martensitic steels to reduce residual activity responses further.

  10. Neural activation during response competition

    NASA Technical Reports Server (NTRS)

    Hazeltine, E.; Poldrack, R.; Gabrieli, J. D.

    2000-01-01

    The flanker task, introduced by Eriksen and Eriksen [Eriksen, B. A., & Eriksen, C. W. (1974). Effects of noise letters upon the identification of a target letter in a nonsearch task. Perception & Psychophysics, 16, 143--149], provides a means to selectively manipulate the presence or absence of response competition while keeping other task demands constant. We measured brain activity using functional magnetic resonance imaging (fMRI) during performance of the flanker task. In accordance with previous behavioral studies, trials in which the flanking stimuli indicated a different response than the central stimulus were performed significantly more slowly than trials in which all the stimuli indicated the same response. This reaction time effect was accompanied by increases in activity in four regions: the right ventrolateral prefrontal cortex, the supplementary motor area, the left superior parietal lobe, and the left anterior parietal cortex. The increases were not due to changes in stimulus complexity or the need to overcome previously learned associations between stimuli and responses. Correspondences between this study and other experiments manipulating response interference suggest that the frontal foci may be related to response inhibition processes whereas the posterior foci may be related to the activation of representations of the inappropriate responses.

  11. Active site specificity of plasmepsin II.

    PubMed Central

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

    1999-01-01

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

  12. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

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

  13. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

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

  14. Updated Site Response Analyses for the Waste Treatment Plant, DOE Hanford, Site, Washington.

    SciTech Connect

    Youngs, Robert R.

    2007-06-29

    This document describes the calculations performed to develop updated relative amplification functions for the Waste Treatment and Immobilization Plant (WTP) facility at the DOE Hanford Site, Washington State. The original 2,000-year return period design spectra for the WTP were based on the results of a probabilistic seismic hazard analysis (PSHA) performed for the DOE Hanford Site by Geomatrix (1996). Geomatrix (1996) performed the PSHA using empirical soil-site ground motion models based primarily on recordings from California. As part of that study, site response analyses were performed to evaluate ground motions at the Hanford sites and California deep soil sites. As described in Appendix A of Geomatrix (1996), characteristic site profiles and dynamic soil properties representative of conditions at various Hanford sites and California deep soil strong motion recording stations were defined. Relative site responses of the Hanford profiles and California profiles were then compared. Based on the results of those site response analyses, it was concluded that ground motions at the Hanford sites underlain by deep soil deposits are similar in character to those on California deep soil sites and it was judged appropriate to use empirical deep soil site attenuation relationships based primarily on California ground motion data to develop design spectra for the Hanford sites. In a subsequent analysis, Geomatrix (2003) updated the site response analyses of Geomatrix (1996, Appendix A) to incorporate randomization of the California and Hanford profiles. The results of that analysis also led to the conclusion that the response of the Hanford profiles was similar to the response of deep soil sites in California.

  15. Updated Site Response Analyses for the Waste Treatment Plant, DOE Hanford Site, Washington

    SciTech Connect

    Youngs RR

    2007-06-01

    This document describes the calculations performed to develop updated relative amplification functions for the Waste Treatment and Immobilization Plant (WTP) facility at the DOE Hanford Site, Washington State. The original 2,000-year return period design spectra for the WTP were based on the results of a probabilistic seismic hazard analysis (PSHA) performed for the DOE Hanford Site by Geomatrix (1996). Geomatrix (1996) performed the PSHA using empirical soil-site ground motion models based primarily on recordings from California. As part of that study, site response analyses were performed to evaluate ground motions at the Hanford sites and California deep soil sites. As described in Appendix A of Geomatrix (1996), characteristic site profiles and dynamic soil properties representative of conditions at various Hanford sites and California deep soil strong motion recording stations were defined. Relative site responses of the Hanford profiles and California profiles were then compared. Based on the results of those site response analyses, it was concluded that ground motions at the Hanford sites underlain by deep soil deposits are similar in character to those on California deep soil sites and it was judged appropriate to use empirical deep soil site attenuation relationships based primarily on California ground motion data to develop design spectra for the Hanford sites. In a subsequent analysis, Geomatrix (2003) updated the site response analyses of Geomatrix (1996, Appendix A) to incorporate randomization of the California and Hanford profiles. The results of that analysis also led to the conclusion that the response of the Hanford profiles was similar to the response of deep soil sites in California.

  16. Environmental Response to Remedial Actions at the Weldon Spring Site--An Environmental Success Story

    SciTech Connect

    Meier, J. A.; Welton, T. D.

    2002-02-27

    Environmental remediation activities have been ongoing at the Weldon Spring Site for over a decade, beginning with small interim response actions and culminating in completion of surface cleanup as represented by closure of the 17 hectare (42-acre) on-site disposal cell. As remedial actions have incrementally been accomplished, the occurrence of site-related contaminants in on and off-site environmental media have effectively been reduced. The DOE-WSSRAP has demonstrated success through the effective reduction or elimination of site related water and airborne contaminants along multiple migration pathways. This paper briefly describes the remedial measures affected at Weldon Spring, and quantifies the environmental responses to those remedial measures.

  17. Responsive service at West Kent sites.

    PubMed

    Baillie, Jonathan

    2011-08-01

    Operations manager, Ashley Heming, and area sales manager, Andy Cassie, of specialist cleaning and cleaning services company, CK Group, explain how a close 15-year relationship with Kent's Maidstone and Tunbridge Wells NHS Trust is continuing, with the firm having recently undertaken a thorough preoccupation clean at the new 513-bed PFI-funded Pembury Hospital, one of the UK's first acute hospitals to offer 100% single-bed en suite accommodation, before its first patients moved in. As HEJ editor Jonathan Baillie explains, the company will undertake similar work for further phases, and has also provided a specialist decontamination service on Victorian buildings set for demolition at the site. PMID:21859067

  18. Measurements of thermospheric response to auroral activities

    NASA Technical Reports Server (NTRS)

    Okano, S.; Kim, J. S.

    1986-01-01

    The Joule heating produced by auroral electrojets and its thermospheric response can be studied by monitoring the thermospheric temperatures by optical methods; simultaneously, the concurrent auroral electrojet activities can be investigated by using geomagnetic records obtained from stations along a meridian close to the observation site of optical measurements. The measurements are reported of thermospheric response to auroral activities which were made at Albany (42.68 deg N, 73.82 deg W), New York on September 2, 1978 (UT) when an isolated substorm occured. The thermospheric temperatures were measured by using a high resolution Fabry-Perot interferometer that determines the line profiles of the (OI) 6300A line emission. The intensities and latitudinal positions of auroral electrojets were obtained by the analysis of magnetograms from the IMS Fort Churchill meridian chain stations.

  19. 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.

  20. Potential sites of CFTR activation by tyrosine kinases.

    PubMed

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

    2016-05-01

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

  1. 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.

  2. Active Response Gravity Offload System

    NASA Technical Reports Server (NTRS)

    Valle, Paul; Dungan, Larry; Cunningham, Thomas; Lieberman, Asher; Poncia, Dina

    2011-01-01

    The Active Response Gravity Offload System (ARGOS) provides the ability to simulate with one system the gravity effect of planets, moons, comets, asteroids, and microgravity, where the gravity is less than Earth fs gravity. The system works by providing a constant force offload through an overhead hoist system and horizontal motion through a rail and trolley system. The facility covers a 20 by 40-ft (approximately equals 6.1 by 12.2m) horizontal area with 15 ft (approximately equals4.6 m) of lifting vertical range.

  3. Nonlinear site response in medium magnitude earthquakes near Parkfield, California

    USGS Publications Warehouse

    Rubinstein, Justin L.

    2011-01-01

    Careful analysis of strong-motion recordings of 13 medium magnitude earthquakes (3.7 ≤ M ≤ 6.5) in the Parkfield, California, area shows that very modest levels of shaking (approximately 3.5% of the acceleration of gravity) can produce observable changes in site response. Specifically, I observe a drop and subsequent recovery of the resonant frequency at sites that are part of the USGS Parkfield dense seismograph array (UPSAR) and Turkey Flat array. While further work is necessary to fully eliminate other models, given that these frequency shifts correlate with the strength of shaking at the Turkey Flat array and only appear for the strongest shaking levels at UPSAR, the most plausible explanation for them is that they are a result of nonlinear site response. Assuming this to be true, the observation of nonlinear site response in small (M M 6.5 San Simeon earthquake and the 2004 M 6 Parkfield earthquake).

  4. An Approach to Identify Site Response Directivity of Accelerometer Sites and Application to the Iranian Area

    NASA Astrophysics Data System (ADS)

    Del Gaudio, Vincenzo; Pierri, Pierpaolo; Rajabi, Ali M.

    2015-06-01

    In recent years, several workers have found numerous cases of sites characterised by significant azimuthal variation of dynamic response to seismic shaking. The causes of this phenomenon are still unclear, but are possibly related to combinations of geological and geomorphological factors determining a polarisation of resonance effects. To improve their comprehension, it would be desirable to extend the database of observations on this phenomenon. Thus, considering that unrevealed cases of site response directivity can be "hidden" among the sites of accelerometer networks, we developed a two-stage approach of data mining from existing strong motion databases to identify sites affected by directional amplification. The proposed procedure first calculates Arias Intensity tensor components from accelerometer recordings of each site to determine mean directional variations of total shaking energy. Then, at the sites where a significant anisotropy appears in ground motion, azimuthal variations of HVSR values (spectral ratios between horizontal and vertical components of recordings) are analysed to confirm the occurrence of site resonance conditions. We applied this technique to a database of recordings acquired by accelerometer stations in the Iranian area. The results of this investigation pointed out some sites affected by directional resonance that appear to be correlated to the orientation of local tectonic lineaments, these being mostly transversal to the direction of maximum shaking. Comparing Arias Intensities observed at these sites with theoretical estimates provided by ground motion prediction equations, the presence of significant site amplifications was confirmed. The magnitude of the amplification factors appear to be correlated to the results of HVSR analysis, even though the pattern of dispersion of HVSR values suggests that while high peak values of spectral ratios are indicative of strong amplifications, lower values do not necessarily imply lower

  5. A Site Response Map of the Continental U.S

    NASA Astrophysics Data System (ADS)

    Magistrale, H.; Rong, Y.; Thompson, E.; Silva, W. J.

    2012-12-01

    We create a site response map of the continental U.S. using the topographic slope methodology, which uses correlations between slope and Vs30 (Wald and Allen, 2007). We determine new regional correlations for the central and eastern U.S. (CEUS) and western U.S. (WUS) calibrated to the Vs30 observation database of Pacific Engineering and Analysis, and introduce a novel correlation for areas of the WUS that hosted Pleistocene and younger lakes. In the WUS we develop a new correlation by first removing Vs30 observations from Utah basins and the Imperial Valley, California, from the database, and second using a stochastic simulation to choose slope and Vs30 bins and calculate standard deviation and bias. We select the best model based on three criteria: (i) bias and standard deviation is among the smallest; (ii) the correlations will not change dramatically for neighboring bins; and (iii) the Vs30 bin boundaries can be matched to NEHRP categories. Relative to WUS, the Utah and Imperial Valley Vs30 values are low for a given slope. We fit a separate correlation in the manner described above. We attribute the low values to the occupation of these areas by Pleistocene and younger lakes. We posit that when sediment-laden streams entered these lakes, the flow velocity was reduced so that all coarse sediments were deposited at the lake margin, and only very fine grained (and seismically slow) material was distributed over the lake bed. This model is confirmed by: (i) relatively high Vs30 values in the geologically similar Las Vegas Valley that was not occupied by a lake; (ii) ordinary Vs30 values in Utah and Imperial Valley locations above the high lake stands; and (iii) a consistent pattern of Vs30 values in the Reno, Nevada, basin across a paleo-lake boundary. In the CEUS, we use the recent correlation of Silva et al. (2011) that includes better constraints on the correlation at rock sites. In all regions the slopes are determined from SRTM digital elevation data (Jarvis

  6. 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?

  7. 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.

  8. 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.

  9. 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

  10. Site Response in the San Joaquin/Sacramento River Delta

    NASA Astrophysics Data System (ADS)

    Fletcher, J. B.; Boatwright, J.

    2007-12-01

    The Sacramento/San Joaquin River Delta lies on the western edge of the Great Valley and contains a system of levees that are thought to be prone to catastrophic failure from a major earthquake in the San Francisco Bay area or on faults along the western border of the Great Valley. To assess this risk we deployed digital recorders and broadband sensors in late 2006 and 2007 at 3 levee sites in the Delta (each site had a top and base sensor) and at one reference site to the west. Cone penetrometer data show that at the base, the soils have low S-wave velocities of 170 to 240 m/s. Upper soil layers are typically peats and aeolian sands. During the nine months of deployment, we recorded 3 local events (45kmSite response is estimated by ratios of S wave spectra at each site to spectra from a reference site to the west of the Delta (Byron Hot Springs , BYR) or Black Diamond Mine (BDM, part of the Berkeley Digital Seismic Network). Spectra are normalized for distance. Each spectrum is smoothed with an algorithm that tries to preserve peaks; a running mean filter is also applied to the spectra from the reference site to reduce the possibility of holes in the reference spectrum appearing as a resonance in the ratio. Our primary observation is that many of the spectral ratios show large resonances, typically at 1-3 Hz and may represent a substantial risk to the levees. Sites at the tops of levees typically have stronger resonances in the 1-3 Hz range compared to base sites. The character of these ratios, however, differs substantially for each event. For example, the top site at Bethel Isl. has peaks in the site response with amplitudes between 6 and 15 (2-3Hz) for an earthquake located near Berkeley using either reference site, but is only apparent in the ratios using BDM for the other two events. This is because BYR has more amplitude in the 2- 3

  11. 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.

  12. A study on the flexibility of enzyme active sites

    PubMed Central

    2011-01-01

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

  13. 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.

  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. Site-specific emergency response concept plans for the Chemical Stockpile Disposal Program

    SciTech Connect

    Carnes, S.A.

    1989-12-01

    Site-specific emergency response concept plans were developed to help initiate enhanced emergency preparedness for continued storage of the stockpile and the Chemical Stockpile Disposal Program (CSDP) at the eight army installations storing the unitary chemical stockpile -- Aberdeen Proving Ground, Anniston Army Depot, Lexington-Blue Grass Army Depot, Newport Army Ammunition Plant, Pine Bluff Arsenal, Pueblo Depot Activity, Tooele Army Depot, and Umatilla Depot Activity. This document summarizes the emergency response plans for all the sites and highlights similarities and differences among them. Section 2 summarizes site-specific differences in stockpile hazard and risk by showing differences in planning-basis accident categories and distributions of topographical features, meteorological conditions, and populations at risk. Section 3 presents a summary of the methodology used to identify the emergency planning zones for each site and the actual recommended boundaries of those zones for the eight sites. Section 4 identifies feasible and recommended protective actions for the sites and explains reasons for differences in them. Finally, Section 5 notes the dependence of protective action effectiveness on the development and implementation of command and control and warning systems that can be implemented in a timely manner, it also identifies the differences in recommended lead times (i.e., from the onset of an accidental release) needed at the sites for effective implementation of protective actions. 17 refs., 11 figs. , 12 tabs.

  16. 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

  17. 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

  18. Site Response and Liquefaction Risk Analysis for Bucharest, Romania

    NASA Astrophysics Data System (ADS)

    Hannich, D.; Ehret, D.; Hoetzl, H.; Grandas, C.; Huber, G.; Bala, A.

    2007-12-01

    Bucharest, the capital of Romania, with more than 2 million inhabitants, is considered, after Istanbul, the second- most earthquake-endangered metropolis in Europe. Four major earthquakes with moment-magnitudes between 6.9 and 7.7 hit Bucharest in the last 65 years. All disastrous earthquakes are generated within a small epicentral area - the Vrancea region - about 150 km northeast of Bucharest. Thick unconsolidated sedimentary layers in the area of Bucharest amplify the arriving seismic shear waves causing severe destruction. Thus, pertinent site response analysis combined with liquefaction risk analysis for the city area are of highest priority for the disaster prevention and mitigation of earthquake effects. Within the frame of the Collaborative Research Center (CRC) 461: "Strong Earthquakes: A Challenge for Geosciences and Civil Engineering, at the University of Karlsruhe, Germany, recently detailed field investigations for the near-surface soil layers in Bucharest were performed. These include Seismic Cone Penetration Tests (SCPTU) and seismic refraction measurements for shallow depths. SCPTU is used to obtain a detailed distribution of the shear wave velocities and in situ state parameters of soils. The results are used for site response analysis with linear and non-linear wave-propagation models as well as for a liquefaction risk analysis. Commonly linear-equivalent models are used to simulate the response of the soil during earthquakes. Nevertheless, the linear-equivalent model cannot reproduce non-linear effects like liquefaction, layer isolation, and consolidation during shaking. Thus, a 1-D wave propagation model was used to study the influence of non- linear effects in the site response analysis. Cone resistance, sleeve friction and pore water pressure registered continuous by the SCPTU method until depths of 35m permit to determine by simplified empirical methods the factor of safety and the probability of liquefaction for different soil layers at

  19. Soil Characterization and Site Response of Marine and Continental Environments

    NASA Astrophysics Data System (ADS)

    Contreras-Porras, R. S.; Huerta-Lopez, C. I.; Martinez-Cruzado, J. A.; Gaherty, J. B.; Collins, J. A.

    2009-05-01

    An in situ soil properties study was conducted to characterize both site and shallow layer sediments under marine and continental environments. Data from the SCoOBA (Sea of Cortez Ocean Bottom Array) seismic experiment and in land ambient vibration measurements on the urban areas of Tijuana, B. C., and Ensenada, B. C., Mexico were used in the analysis. The goal of this investigation is to identify and to analyze the effect of the physical/geotechnical properties of the ground on the site response upon seismic excitations in both marine and continental environments. The time series were earthquakes and background noise recorded within interval of 10/2005 to 10/2006 in the Gulf of California (GoC) with very-broadband Ocean Bottom Seismographs (OBS), and ambient vibration measurements collected during different time periods on Tijuana and Ensenada urban areas. The data processing and analysis was conducted by means of the H/V Spectral Ratios (HVSPR) of multi component data, the Random Decrement Method (RDM), and Blind Deconvolution (BD). This study presents ongoing results of a long term project to characterize the local site response of soil layers upon dynamic excitations using digital signal processing algorithms on time series, as well as the comparison between the results these methodologies are providing.

  20. A geostatistical approach to mapping site response spectral amplifications

    USGS Publications Warehouse

    Thompson, E.M.; Baise, L.G.; Kayen, R.E.; Tanaka, Y.; Tanaka, H.

    2010-01-01

    If quantitative estimates of the seismic properties do not exist at a location of interest then the site response spectral amplifications must be estimated from data collected at other locations. Currently, the most common approach employs correlations of site class with maps of surficial geology. Analogously, correlations of site class with topographic slope can be employed where the surficial geology is unknown. Our goal is to identify and validate a method to estimate site response with greater spatial resolution and accuracy for regions where additional effort is warranted. This method consists of three components: region-specific data collection, a spatial model for interpolating seismic properties, and a theoretical method for computing spectral amplifications from the interpolated seismic properties. We consider three spatial interpolation schemes: correlations with surficial geology, termed the geologic trend (GT), ordinary kriging (OK), and kriging with a trend (KT). We estimate the spectral amplifications from seismic properties using the square root of impedance method, thereby linking the frequency-dependent spectral amplifications to the depth-dependent seismic properties. Thus, the range of periods for which this method is applicable is limited by the depth of exploration. A dense survey of near-surface S-wave slowness (Ss) throughout Kobe, Japan shows that the geostatistical methods give more accurate estimates of Ss than the topographic slope and GT methods, and the OK and KT methods perform equally well. We prefer the KT model because it can be seamlessly integrated with geologic maps that cover larger regions. Empirical spectral amplifications show that the region-specific data achieve more accurate estimates of observed median short-period amplifications than the topographic slope method. ?? 2010 Elsevier B.V.

  1. SAVANNAH RIVER SITE CAPABILITIES FOR CONDUCTING INGESTION PATHWAY CONSEQUENCE ASSESSMENTS FOR EMERGENCY RESPONSE

    SciTech Connect

    Hunter, C

    2007-12-11

    Potential airborne releases of radioactivity from facilities operated for the U. S. Department of Energy at the Savannah River Site could pose significant consequences to the public through the ingestion pathway. The Savannah River National Laboratory has developed a suite of technologies needed to conduct assessments of ingestion dose during emergency response, enabling emergency manager at SRS to develop initial protective action recommendation for state agencies early in the response and to make informed decisions on activation of additional Federal assets that would be needed to support long-term monitoring and assessment activities.

  2. Active Sites Environmental Monitoring Program FY 1996 annual report

    SciTech Connect

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

    1997-11-01

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

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

    SciTech Connect

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

    1994-05-01

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

  4. Amchitka Island, Alaska, Potential U.S. Department of Energy Site Responsibilities

    SciTech Connect

    U.S. Department of Energy, Nevada Operations Office

    1999-01-22

    This historical records review report concerns the activities of the US Atomic Energy Commission (AEC) at Amchitka Island, Alaska, over a period extending from 1942 to 1993. The report focuses on AEC activities resulting in known or suspected contamination of the island environment by nonradiological hazardous or toxic materials as discerned through historical records. In addition, the information from historical records was augmented by an August 1998 sampling event. Both the records review and sampling were conducted by IT Corporation on behalf of the US Department of Energy (DOE), the predecessor agency to the AEC. The intent of this investigation was to identify all potentially contaminated sites for which DOE may be responsible, wholly or partially, including all official sites of concern as recognized by the US Fish and Wildlife Service (USFWS). Additionally, potential data gaps that the DOE will need to fill to support the ecological and human health risk assessments performed were identified. A review of the available historical information regarding AEC's activities on Amchitka Island indicates that the DOE is potentially responsible for 11 sites identified by USFWS and an additional 10 sites that are not included in the USFWS database of sites of potential concern.

  5. Source Spectra and Site Response for Two Indonesian Earthquakes: the Tasikmalaya and Kerinci Events of 2009

    NASA Astrophysics Data System (ADS)

    Gunawan, I.; Cummins, P. R.; Ghasemi, H.; Suhardjono, S.

    2012-12-01

    Indonesia is very prone to natural disasters, especially earthquakes, due to its location in a tectonically active region. In September-October 2009 alone, intraslab and crustal earthquakes caused the deaths of thousands of people, severe infrastructure destruction and considerable economic loss. Thus, both intraslab and crustal earthquakes are important sources of earthquake hazard in Indonesia. Analysis of response spectra for these intraslab and crustal earthquakes are needed to yield more detail about earthquake properties. For both types of earthquakes, we have analysed available Indonesian seismic waveform data to constrain source and path parameters - i.e., low frequency spectral level, Q, and corner frequency - at reference stations that appear to be little influenced by site response.. We have considered these analyses for the main shocks as well as several aftershocks. We obtain corner frequencies that are reasonably consistent with the constant stress drop hypothesis. Using these results, we consider using them to extract information about site response form other stations form the Indonesian strong motion network that appear to be strongly affected by site response. Such site response data, as well as earthquake source parameters, are important for assessing earthquake hazard in Indonesia.

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

    PubMed

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

    2016-06-17

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

  7. Hanford Site emergency response needs, Volumes 1 and 2

    SciTech Connect

    Good, D.E.

    1996-04-16

    This report presents the results of a comprehensive third party needs assessment of the Hanford Fire Department (HFD), conducted by Hughes Associates Inc. The assessment was commissioned with the intent of obtaining an unbiased report which could be used as a basis for identifying needed changes/modifications to the fire department and its services. This report serves several functions: (1) it documents current and future site operations and associated hazards and risks identified as a result of document review, site and facility surveys, and interviews with knowledgeable personnel; (2) describes the HFD in terms of organization, existing resources and response capabilities; (3) identifies regulatory and other requirements that are applicable to the HFD and includes a discussion of associated legal liabilities; and (4) provides recommendations based on applicable requirements and existing conditions. Each recommendation is followed by a supporting statement to clarify the intent or justification of the recommendation. This report will be followed by a Master Plan document which will present an implementation method for the recommendations (with associated costs) considered to be essential to maintaining adequate, cost effective emergency services at the Hanford site in the next five to seven years.

  8. 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.

  9. CheY’s acetylation sites responsible for generating clockwise flagellar rotation in Escherichia coli

    PubMed Central

    Fraiberg, Milana; Afanzar, Oshri; Cassidy, C. Keith; Gabashvili, Alexandra; Schulten, Klaus; Levin, Yishai; Eisenbach, Michael

    2015-01-01

    Summary Stimulation of Escherichia coli with acetate elevates the acetylation level of the chemotaxis response regulator CheY. This elevation, in an unknown mechanism, activates CheY to generate clockwise rotation. Here, using quantitative selective reaction monitoring mass spectrometry and high-resolution targeted mass spectrometry, we identified K91 and K109 as the major sites whose acetylation level in vivo increases in response to acetate. Employing single and multiple lysine replacements in CheY, we found that K91 and K109 are also the sites mainly responsible for acetate-dependent clockwise generation. Furthermore, we showed that clockwise rotation is repressed when residue K91 is non-modified, as evidenced by an increased ability of CheY to generate clockwise rotation when K91 was acetylated or replaced by specific amino acids. Using molecular dynamics simulations we show that K91 repression is manifested in the conformational dynamics of the β4α4 loop, shifted towards an active state upon mutation. Removal of β4α4 loop repression may represent a general activation mechanism in CheY, pertaining also to the canonical phosphorylation activation pathway as suggested by crystal structures of active and inactive CheY from Thermotoga maritima. By way of elimination we further suggest that K109 acetylation is actively involved in generating clockwise rotation. PMID:25388160

  10. Productivity, mortality, and response to disturbance of nesting Swainson's hawks on the Hanford Site

    SciTech Connect

    Poole, L.D.; Marr, N.V.; McCorquodale, S.M.

    1988-03-01

    The objectives of this study were to characterize Swainson's hawk (Buteo swainson) use of the US Department of Energy (DOE) Hanford Site and to evaluate the potential for engineering and other human activities on the Hanford Site to negatively affect the nesting Swainson's hawk population. Activities associated with the Basalt Waste Isolation Project (BWIP) were used as the primary external stimuli in studying hawk responses to potential human disturbance. Parked and moving vehicles were the most common disturbance sources observed in Swainson's hawk territories. Hawks appeared to be sensitive to disturbance from pedestrians and slow-moving vehicles near nests. Novel stimuli were much more likely to evoke strong responses than were recurring events. Adult hawks reacted more frequently and vigorously than did juveniles. When disturbed, adult hawks usually flew toward the location of the disturbance; juvenile hawks usually flew away from disturbances. Human activity associated with BWIP may have had negative on one pair of nesting Swainson's hawks and may have precluded the use of an additional traditional nesting territory. Negative impacts to nesting Swainson's hawks from human activity could be minimized by confining activities to the non-nesting period or to distances greater than 2.2 km from nest sites. Tree groves and elevated perches, including utility poles, across the Hanford Site are probably critical to the success of nesting Swainson's hawks. Potential mitigation strategies associated with energy research and development activities on the Hanford Site could include provisions for maintenance and establishment of drought-tolerant trees and native vegetation. 22 refs., 5 figs., 3 tabs.

  11. Physiological Response to Physical Activity in Children.

    ERIC Educational Resources Information Center

    Gilliam, Thomas B.

    This is a report on research in the field of physical responses of children to strenuous activity. The paper is divided into three subtopics: (1) peak performance measure in children; (2) training effects on children; and (3) importance of physical activity for children. Measurements used are oxygen consumption, ventilation, heart rate, cardiac…

  12. 77 FR 31010 - Proposed CERCLA Agreement for Recovery of Past Response Costs; Piqua Hospital Site

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-24

    ... AGENCY Proposed CERCLA Agreement for Recovery of Past Response Costs; Piqua Hospital Site AGENCY... of past response costs concerning the Piqua Hospital Site (Site ID Number B5RB) in Piqua, Ohio with...: 312-353-6121. Comments should reference the Piqua Hospital Site in Piqua, Ohio and EPA Docket No....

  13. Dashboard applications to monitor experiment activities at sites

    NASA Astrophysics Data System (ADS)

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

    2010-04-01

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

  14. 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

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

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

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

  16. 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

  17. Stress Reorganization and Response in Active Solids

    NASA Astrophysics Data System (ADS)

    Hawkins, Rhoda J.; Liverpool, Tanniemola B.

    2014-07-01

    We present a microscopic model of a disordered viscoelastic active solid, i.e., an active material whose long time behavior is elastic as opposed to viscous. It is composed of filaments, passive cross-links, and molecular motors powered by stored chemical energy, e.g., actomyosin powered by adenosine triphosphate. Our model allows us to study the collective behavior of contractile active elements and how their interaction with each other and the passive elastic elements determines the macroscopic mechanical properties of the active material. As a result of the (un)binding dynamics of the active elements, we find that this system provides a highly responsive material with a dynamic mechanical response strongly dependent on the amount of deformation.

  18. 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.

  19. Molecular Imprint of Enzyme Active Site by Camel Nanobodies

    PubMed Central

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

    2012-01-01

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

  20. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Contracts for Superfund Response Actions Combining Cooperative Agreements § 35.6260 Combining...

  1. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 1 2014-07-01 2014-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  2. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 1 2011-07-01 2011-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  3. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 1 2013-07-01 2013-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  4. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 1 2012-07-01 2012-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response...

  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. An active-site peptide from pepsin C

    PubMed Central

    Kay, J.; Ryle, A. P.

    1971-01-01

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

  7. Analysis of ground response data at Lotung large-scale soil- structure interaction experiment site

    SciTech Connect

    Chang, C.Y.; Mok, C.M.; Power, M.S. )

    1991-12-01

    The Electric Power Research Institute (EPRI), in cooperation with the Taiwan Power Company (TPC), constructed two models (1/4-scale and 1/2-scale) of a nuclear plant containment structure at a site in Lotung (Tang, 1987), a seismically active region in northeast Taiwan. The models were constructed to gather data for the evaluation and validation of soil-structure interaction (SSI) analysis methodologies. Extensive instrumentation was deployed to record both structural and ground responses at the site during earthquakes. The experiment is generally referred to as the Lotung Large-Scale Seismic Test (LSST). As part of the LSST, two downhole arrays were installed at the site to record ground motions at depths as well as at the ground surface. Structural response and ground response have been recorded for a number of earthquakes (i.e. a total of 18 earthquakes in the period of October 1985 through November 1986) at the LSST site since the completion of the installation of the downhole instruments in October 1985. These data include those from earthquakes having magnitudes ranging from M{sub L} 4.5 to M{sub L} 7.0 and epicentral distances range from 4.7 km to 77.7 km. Peak ground surface accelerations range from 0.03 g to 0.21 g for the horizontal component and from 0.01 g to 0.20 g for the vertical component. The objectives of the study were: (1) to obtain empirical data on variations of earthquake ground motion with depth; (2) to examine field evidence of nonlinear soil response due to earthquake shaking and to determine the degree of soil nonlinearity; (3) to assess the ability of ground response analysis techniques including techniques to approximate nonlinear soil response to estimate ground motions due to earthquake shaking; and (4) to analyze earth pressures recorded beneath the basemat and on the side wall of the 1/4 scale model structure during selected earthquakes.

  8. 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

  9. Active thermal isolation for temperature responsive sensors

    NASA Technical Reports Server (NTRS)

    Martinson, Scott D. (Inventor); Gray, David L. (Inventor); Carraway, Debra L. (Inventor); Reda, Daniel C. (Inventor)

    1994-01-01

    A temperature responsive sensor is located in the airflow over the specified surface of a body and is maintained at a constant temperature. An active thermal isolator is located between this temperature responsive sensor and the specified surface of the body. The temperature of this isolator is controlled to reduce conductive heat flow from the temperature responsive sensor to the body. This temperature control includes: (1) operating the isolator at the same temperature as the constant temperature of the sensor and (2) establishing a fixed boundary temperature which is either less than or equal to or slightly greater than the sensor constant temperature.

  10. Cellular Stress Responses and Monitored Cellular Activities.

    PubMed

    Sawa, Teiji; Naito, Yoshifumi; Kato, Hideya; Amaya, Fumimasa

    2016-08-01

    To survive, organisms require mechanisms that enable them to sense changes in the outside environment, introduce necessary responses, and resist unfavorable distortion. Consequently, through evolutionary adaptation, cells have become equipped with the apparatus required to monitor their fundamental intracellular processes and the mechanisms needed to try to offset malfunction without receiving any direct signals from the outside environment. It has been shown recently that eukaryotic cells are equipped with a special mechanism that monitors their fundamental cellular functions and that some pathogenic proteobacteria can override this monitoring mechanism to cause harm. The monitored cellular activities involved in the stressed intracellular response have been researched extensively in Caenorhabditis elegans, where discovery of an association between key mitochondrial activities and innate immune responses was named "cellular associated detoxification and defenses (cSADD)." This cellular surveillance pathway (cSADD) oversees core cellular activities such as mitochondrial respiration and protein transport into mitochondria, detects xenobiotics and invading pathogens, and activates the endocrine pathways controlling behavior, detoxification, and immunity. The cSADD pathway is probably associated with cellular responses to stress in human inflammatory diseases. In the critical care field, the pathogenesis of lethal inflammatory syndromes (e.g., respiratory distress syndromes and sepsis) involves the disturbance of mitochondrial respiration leading to cell death. Up-to-date knowledge about monitored cellular activities and cSADD, especially focusing on mitochondrial involvement, can probably help fill a knowledge gap regarding the pathogenesis of lethal inflammatory syndromes in the critical care field. PMID:26954943

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

    PubMed

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

    1987-11-01

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

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

    PubMed Central

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

    2015-01-01

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

  13. 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.

  14. Teaching Responsibility through Physical Activity. Second Edition.

    ERIC Educational Resources Information Center

    Hellison, Don

    This book guides teachers in using physical activity to foster personal and social responsibility. Focusing on teaching in school settings, the book features comments from real students to motivate teachers to apply the concept; take-aways that summarize each chapter and help teachers consider their own situations; new chapters on the lesson plan…

  15. Rigor and Responsiveness in Classroom Activity

    ERIC Educational Resources Information Center

    Thomspon, Jessica; Hagenah, Sara; Kang, Hosun; Stroupe, David; Braaten, Melissa; Colley, Carolyn; Windschitl, Mark

    2016-01-01

    Background/Context: There are few examples from classrooms or the literature that provide a clear vision of teaching that simultaneously promotes rigorous disciplinary activity and is responsive to all students. Maintaining rigorous and equitable classroom discourse is a worthy goal, yet there is no clear consensus of how this actually works in a…

  16. 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

  17. Localization of the binding site of tissue-type plasminogen activator to fibrin.

    PubMed Central

    Ichinose, A; Takio, K; Fujikawa, K

    1986-01-01

    Functionally active A and B chains were separated from a two-chain form of recombinant tissue-type plasminogen activator after mild reduction and alkylation. The A chain was found to be responsible for the binding to lysine-Sepharose or fibrin and the B chain contained the catalytic activity of tissue-type plasminogen activator. An extensive reduction of two-chain tissue-type plasminogen activator, however, destroyed both the binding and catalytic activities. A thermolytic fragment, Fr. 1, of tissue-type plasminogen activator that contained a growth factor and two kringle segments retained its lysine binding activity. Additional thermolytic cleavages in the kringle-2 segment of Fr. 1 caused a total loss of the binding activity. These results indicated that the binding site of tissue-type plasminogen activator to fibrin was located in the kringle-2 segment. Images PMID:3088041

  18. 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.

  19. 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…

  20. Limitation and applicability of microtremor records for site-response estimation

    NASA Astrophysics Data System (ADS)

    Song, G.; Kang, T.; Park, S.

    2010-12-01

    Site effects are the modifications of seismic motions which are traveling through near-surface materials. The impedance contrast between the topmost layer and bedrock may significantly amplify ground motions and augment their durations. Inelastic behavior of the geological media such as highly fractured/weathered rocks and unconsolidated sediments may absorb seismic energy, and thus damp the resulting ground motions. It is inherently most desirable to evaluate the site effects using seismic records from large earthquakes. If there are only small events that will be recorded by several seismograph stations, it becomes difficult to evaluate site effects using earthquake data. Recently a number of studies pay attention to microtremor records to assess site effects. The main reason of such efforts is that measurements are relatively easy regardless of site condition and cost-effective without necessity of waiting for earthquakes or of using active sources. Especially microtremor measurements are exclusively a useful option to assess site effects, and thus seismic microzonation, in the urban area and/or region of low to moderate seismicity. Spectral ratios of horizontal components to vertical component (HVSR) of microtremor records have been popular for estimation of site resonant frequency. Although some studies have shown that the amplitude of spectral ratios is an indicator of site amplification relative to bedrock motion, there are still debates on it. This discrepancy may originate from the deficiency of our understanding on the nature of microtremor. Therefore, it is important to understand the limitation and applicability of microtremor records for site-effect assessments. The focus on this problem is how microtremor responses on the subsurface structures and their physical properties, and how parameters deduced from microtremor analyses are related to site responses during earthquake ground motions. In order to investigate how these issues have a practical

  1. Estimating Derived Response Levels at the Savannah River Site for Use with Emergency Response Models

    SciTech Connect

    Simpkins, A.A.

    2002-12-06

    Emergency response computer models at the Savannah River Site (SRS) are coupled with real-time meteorological data to estimate dose to individuals downwind of accidental radioactive releases. Currently, these models estimate doses for inhalation and shine pathways, but do not consider dose due to ingestion of contaminated food products. The Food and Drug Administration (FDA) has developed derived intervention levels (DIL) which refer to the radionuclide-specific concentration in food present throughout the relevant period of time, with no intervention, that could lead to an individual receiving a radiation dose equal to the protective action guide. In the event of an emergency, concentrations in various food types are compared with these levels to make interdictions decisions. Prior to monitoring results being available, concentrations in the environmental media (i.e. soil), called derived response levels (DRLs), can be estimated from the DILs and directly compared with computer output to provide preliminary guidance as to whether intervention is necessary. Site-specific derived response levels (DRLs) are developed for ingestion pathways pertinent to SRS: milk, meat, fish, grain, produce, and beverage. This provides decision-makers with an additional tool for use immediately following an accident prior to the acquisition of food monitoring data.

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

    SciTech Connect

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

    1994-04-01

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

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

    PubMed

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

    2007-06-26

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

  4. Chemical and biomarker responses for site-specific quality assessment of the Lake Maggiore (Northern Italy).

    PubMed

    Parolini, Marco; Pedriali, Alessandra; Binelli, Andrea

    2013-08-01

    Since the 1990s, the Lake Maggiore (Northern Italy) has been recognized as an aquatic environment contaminated by DDTs and other persistent organic pollutants, but to date just few studies were carried out to investigate the effects of pollution to aquatic organisms. The aim of this study was the application of a stepwise approach based on chemical data, a suite of biomarkers and the integration of their responses into a biomarker response index (BRI) to evaluate the site-specific quality assessment in different sampling stations of Lake Maggiore, one of the largest European lakes. We used as biological model the freshwater bivalve Zebra mussel (Dreissena polymorpha). Several hundred bivalve specimens were sampled on May 2011 from eight sampling sites located along the lake shoreline. We measured levels of DDTs, PCBs, HCHs, HCB, and PAHs accumulated in D. polymorpha soft tissues by GC/MSn, while the activities of catalase, superoxide dismutase, glutathione peroxidase, and glutathione S-transferase, as well as the lipid peroxidation and protein carbonyl content were evaluated in homogenates from native bivalves as oxidative stress indices. Moreover, DNA damage was investigated by the alkaline precipitation assay. Significant imbalances of enzymatic activity were found in mussels from most of the sampling sites, as well as notable increases of damage to macromolecules. Health status of mussels from Baveno was greatly affected by lake pollution, probably due to high levels of DDTs measured in this site, while a wide variability in biomarker responses was found in all the other stations. The application of a BRI allowed distinguishing impacts of pollution to bivalves, confirming mussels from Baveno as the most threatened and revealing that also the health status of bivalves from Suna, Brissago, Pallanza, and Laveno is affected. These evidences suggest the usefulness of a specific index to integrate all the biomarker endpoints in order to provide a correct environmental

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

    PubMed Central

    Petrosino, J F; Palzkill, T

    1996-01-01

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

  6. A PTH-responsive circadian clock operates in ex vivo mouse femur fracture healing site.

    PubMed

    Kunimoto, Tatsuya; Okubo, Naoki; Minami, Yoichi; Fujiwara, Hiroyoshi; Hosokawa, Toshihiro; Asada, Maki; Oda, Ryo; Kubo, Toshikazu; Yagita, Kazuhiro

    2016-01-01

    The circadian clock contains clock genes including Bmal1 and Period2, and it maintains an interval rhythm of approximately 24 hours (the circadian rhythm) in various organs including growth plate and articular cartilage. As endochondral ossification is involved not only in growth plate but also in fracture healing, we investigated the circadian clock functions in fracture sites undergoing healing. Our fracture models using external fixation involved femurs of Period2::Luciferase knock-in mice which enables the monitoring of endogenous circadian clock state via bioluminescence. Organ culture was performed by collecting femurs, and fracture sites were observed using bioluminescence imaging systems. Clear bioluminescence rhythms of 24-hour intervals were revealed in fracture healing sites. When parathyroid hormone (PTH) was administered to fractured femurs in organ culture, peak time of Period2::Luciferase activity in fracture sites and growth plates changed, indicating that PTH-responsive circadian clock functions in the mouse femur fracture healing site. While PTH is widely used in treating osteoporosis, many studies have reported that it contributes to improvement of fracture healing. Future studies of the role of this local clock in wound healing may reveal a novel function of the circadian timing mechanism in skeletal cells. PMID:26926165

  7. A PTH-responsive circadian clock operates in ex vivo mouse femur fracture healing site

    PubMed Central

    Kunimoto, Tatsuya; Okubo, Naoki; Minami, Yoichi; Fujiwara, Hiroyoshi; Hosokawa, Toshihiro; Asada, Maki; Oda, Ryo; Kubo, Toshikazu; Yagita, Kazuhiro

    2016-01-01

    The circadian clock contains clock genes including Bmal1 and Period2, and it maintains an interval rhythm of approximately 24 hours (the circadian rhythm) in various organs including growth plate and articular cartilage. As endochondral ossification is involved not only in growth plate but also in fracture healing, we investigated the circadian clock functions in fracture sites undergoing healing. Our fracture models using external fixation involved femurs of Period2::Luciferase knock-in mice which enables the monitoring of endogenous circadian clock state via bioluminescence. Organ culture was performed by collecting femurs, and fracture sites were observed using bioluminescence imaging systems. Clear bioluminescence rhythms of 24-hour intervals were revealed in fracture healing sites. When parathyroid hormone (PTH) was administered to fractured femurs in organ culture, peak time of Period2::Luciferase activity in fracture sites and growth plates changed, indicating that PTH-responsive circadian clock functions in the mouse femur fracture healing site. While PTH is widely used in treating osteoporosis, many studies have reported that it contributes to improvement of fracture healing. Future studies of the role of this local clock in wound healing may reveal a novel function of the circadian timing mechanism in skeletal cells. PMID:26926165

  8. 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.

  9. 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.

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

    PubMed Central

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

    2000-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  12. Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

    SciTech Connect

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong; Chen, Shoudeng; Zhou, Jie; He, Xiaojing; Lv, Zongyang; Ge, Ruowen; Li, Xuemei; Deng, Tao; Fodor, Ervin; Rao, Zihe; Liu, Yingfang

    2009-11-10

    The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenza H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.

  13. Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity

    PubMed Central

    Rinehart, Jesse; Maksimova, Yelena D.; Tanis, Jessica E.; Stone, Kathryn L.; Hodson, Caleb A.; Zhang, Junhui; Risinger, Mary; Pan, Weijun; Wu, Dianqing; Colangelo, Christopher M.; Forbush, Biff; Joiner, Clinton H.; Gulcicek, Erol E.; Gallagher, Patrick G.; Lifton, Richard P.

    2010-01-01

    Summary Modulation of intracellular chloride concentration ([Cl−]i) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl− exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl−]I; however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl−]i and have implications for control of cell volume and neuronal function. PMID:19665974

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

    SciTech Connect

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

    1994-08-01

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

  15. The unfolded protein response triggers site-specific regulatory ubiquitylation of 40S ribosomal proteins

    PubMed Central

    Rising, Lisa; Mak, Raymond; Webb, Kristofor; Kaiser, Stephen E.; Zuzow, Nathan; Riviere, Paul; Yang, Bing; Fenech, Emma; Tang, Xin; Lindsay, Scott A.; Christianson, John C.; Hampton, Randolph Y.; Wasserman, Steven A.; Bennett, Eric J.

    2015-01-01

    Summary Insults to endoplasmic reticulum (ER) homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as a previously uncharacterized and important facet of eukaryotic translational control. PMID:26051182

  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. ESTIMATION OF RESPONSE-SPECTRAL VALUES AS FUNCTIONS OF MAGNITUDE, DISTANCE, AND SITE CONDITIONS.

    USGS Publications Warehouse

    Joyner, W.B.; Boore, D.M.

    1983-01-01

    Horizontal pseudo-velocity response was analyzed for twelve shallow earthquakes in western North America. Estimation of response-spectral values was related to magnitude, distance and site conditions. Errors in the methods are analyzed.

  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. COSTS OF REMEDIAL RESPONSE ACTIONS AT UNCONTROLLED HAZARDOUS WASTE SITES

    EPA Science Inventory

    The primary purpose of this study was to update conceptual design cost estimates for remedial action unit operations portrayed in earlier reports. Thirty-five remedial action unit operations conceptual designs, addressing uncontrolled landfill or impoundment disposal sites, were ...

  20. The role of biominerals in enhancing the geophysical response at hydrocarbon contaminated sites

    NASA Astrophysics Data System (ADS)

    Mewafy, Farag Mohamed

    There are several source mechanisms by which microbial activity in the subsurface can change geophysical signatures. To date the source mechanisms generating the geophysical signatures in microbially active environments remain poorly understood. In this study, we investigated the link between the biogeochemical processes resulting in biotransformation of metallic iron mineral phases and the associated biogeophysical signatures. Hydrocarbon contaminated environments provide excellent laboratories for investigating iron mineral biotransformation. In particular, we investigated the magnetic susceptibility (MS) and the complex conductivity (CC) signatures of a hydrocarbon contaminated site near Bemidji, Minnesota. For the MS study, we investigated the changes in the MS response for cores retrieved from the site as well as down boreholes. The contaminated location revealed two enriched MS zones. The first MS lies within the hydrocarbon smear zone, which is limited to the zone of water table fluctuation with high concentrations of dissolved Fe(II) and organic carbon content. Magnetite and siderite were the dominant minerals formed during this process. However, magnetite was responsible for the bulk of MS changes. The second zone of MS enhancement lies within the vadose zone which is characterized by methane depletion suggesting that aerobic or anaerobic oxidation of methane is coupled to iron-reduction resulting in magnetite precipitation. For the CC work, we conducted laboratory CC measurements along four cores in addition to field CC survey. We found that the real (sigma‧) and imaginary (sigma″) conductivity are higher for samples from within the oil plume especially within the smear zone compared to background uncontaminated samples. Using magnetite as an example of the biometallic minerals in the smear zone at the site, a clear increase in the sigma″ response with increasing magnetite content was observed suggesting that the presence of bio-metallic mineral

  1. Lessons learned from DOE site culture change activities: Implications for waste management organizations

    SciTech Connect

    Kurstedt, H.A. Jr.; Howard, E.M.; Doss, A.R.; Mallak, L.A.

    1991-01-01

    Management Systems Laboratories (MSL) has worked with the US Department of Energy (DOE) and several of its contractors as they understand and assess the DOE culture change and change the contractor culture to serve DOE's needs. Primarily, these contractors have been those whose responsibilities include starting up and operating weapons materials facilities. The number and scope of these activities have escalated and expanded to contractors at DOE sites such as Westinghouse at the Savannah River Site (SRS) in Aiken, South Carolina, EG G at the Rocky Flats Plant (RFP) in Golden, Colorado, and Westinghouse at the Feed Materials Processing Center (FMPC) in Fernald, Ohio. The point of this paper is not to compare or contrast the relative merit of one site over another. It is to show the lessons, good and bad, and use and communicate those lessons, especially those lessons transferable to other sites in similar situations. 8 refs., 1 fig.

  2. 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.

  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. Active thermal isolation for temperature responsive sensors

    NASA Technical Reports Server (NTRS)

    Martinson, Scott D. (Inventor); Gray, David L. (Inventor); Carraway, Debra L. (Inventor); Reda, Daniel C. (Inventor)

    1994-01-01

    The detection of flow transition between laminar and turbulent flow and of shear stress or skin friction of airfoils is important in basic research for validation of airfoil theory and design. These values are conventionally measured using hot film nickel sensors deposited on a polyimide substrate. The substrate electrically insulates the sensor and underlying airfoil but is prevented from thermally isolating the sensor by thickness constraints necessary to avoid flow contamination. Proposed heating of the model surface is difficult to control, requires significant energy expenditures, and may alter the basic flow state of the airfoil. A temperature responsive sensor is located in the airflow over the specified surface of a body and is maintained at a constant temperature. An active thermal isolator is located between this temperature responsive sensor and the specific surface of the body. The total thickness of the isolator and sensor avoid any contamination of the flow. The temperature of this isolator is controlled to reduce conductive heat flow from the temperature responsive sensor to the body. This temperature control includes (1) operating the isolator at the same temperature as the constant temperature of the sensor; and (2) establishing a fixed boundary temperature which is either less than or equal to, or slightly greater than the sensor constant temperature. The present invention accordingly thermally isolates a temperature responsive sensor in an energy efficient, controllable manner while avoiding any contamination of the flow.

  5. Dynamic response of active twist rotor blades

    NASA Astrophysics Data System (ADS)

    Cesnik, Carlos E. S.; Shin, Sang Joon; Wilbur, Matthew L.

    2001-02-01

    Dynamic characteristics of active twist rotor (ATR) blades are investigated analytically and experimentally in this paper. The ATR system is intended for vibration and potentially for noise reductions in helicopters through individual blade control. An aeroelastic model is developed to identify frequency response characteristics of the ATR blade with integral, generally anisotropic, strain actuators embedded in its composite construction. An ATR prototype blade was designed and manufactured to experimentally study the vibration reduction capabilities of such systems. Several bench and hover tests were conducted and those results are presented and discussed here. Selected results on sensitivity of the ATR system to collective setting (i.e. blade loading), blade rpm (i.e. centrifugal force and blade station velocity), and media density (i.e. altitude) are presented. They indicated that the twist actuation authority of the ATR blade is independent of the collective setting up to approximately 10P, and dependent on rotational speed and altitude near the torsional resonance frequency due to its dependency on the aerodynamic damping. The proposed model captures very well the physics and sensitivities to selected test parameters of the ATR system. The numerical result of the blade torsional loads show an average error of 20% in magnitude and virtually no difference in phase for the blade frequency response. Overall, the active blade model is in very good agreement with the experiments and can be used to analyze and design future active helicopter blade systems.

  6. GAMMA DETECTOR RESPONSE/SOIL CONCENTRATION CORRELATION STUDY AT THE AAR MANUFACTURING, INC. SITE, LIVONIA, MICHIGAN

    SciTech Connect

    ALTIC, NICK A

    2013-03-22

    At the NRC's request, ORAU conducted surveys of the AAR Manufacturing site during the period of September 25 through September 27, 2012. The survey activities included walkover surveys and sampling activities. Once the survey team was onsite, the NRC personnel decided to forgo survey activities in the New Addition and the pickling area. Areas of the planned study boundary were inaccessible due to overgrowth/large pieces of concrete covering the soil surface; therefore, the study boundary was redefined. Gamma walkover scans of the site boundary and front yard identified multiple areas of elevated gamma radiation. As a result, two judgmental samples were collected. Sample results were above thorium background levels The answer to the PSQ relating to the relationship between thorium concentration in soil and NaI instrument response is Yes. NaI instrument response can be used as a predictor of Th-232 concentration in the 0 to 1 m layer. An R2 value of 0.79 was determined for the surface soil relationship, thus satisfying the DQOs. Moreover, the regression was cross-checked by comparing the predicted Th-232 soil core concentration to the average Th-232 concentration (Section 5.3.2). Based on the cross-check, the regression equation provides a reasonable estimate for the Th-232 concentration at the judgmental locations. Consideration must be given when applying this equation to other soil areas of the site. If the contamination was heterogeneously distributed, and not distributed in a discrete layer as it was in the study area, then using the regression equation to predict Th-232 concentration would not be applicable.

  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. 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

  9. Trisomy 21 consistently activates the interferon response

    PubMed Central

    Sullivan, Kelly D; Lewis, Hannah C; Hill, Amanda A; Pandey, Ahwan; Jackson, Leisa P; Cabral, Joseph M; Smith, Keith P; Liggett, L Alexander; Gomez, Eliana B; Galbraith, Matthew D; DeGregori, James; Espinosa, Joaquín M

    2016-01-01

    Although it is clear that trisomy 21 causes Down syndrome, the molecular events acting downstream of the trisomy remain ill defined. Using complementary genomics analyses, we identified the interferon pathway as the major signaling cascade consistently activated by trisomy 21 in human cells. Transcriptome analysis revealed that trisomy 21 activates the interferon transcriptional response in fibroblast and lymphoblastoid cell lines, as well as circulating monocytes and T cells. Trisomy 21 cells show increased induction of interferon-stimulated genes and decreased expression of ribosomal proteins and translation factors. An shRNA screen determined that the interferon-activated kinases JAK1 and TYK2 suppress proliferation of trisomy 21 fibroblasts, and this defect is rescued by pharmacological JAK inhibition. Therefore, we propose that interferon activation, likely via increased gene dosage of the four interferon receptors encoded on chromosome 21, contributes to many of the clinical impacts of trisomy 21, and that interferon antagonists could have therapeutic benefits. DOI: http://dx.doi.org/10.7554/eLife.16220.001 PMID:27472900

  10. 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.

  11. 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.

  12. Dynamics of active cellular response under stress

    NASA Astrophysics Data System (ADS)

    de, Rumi; Zemel, Assaf; Safran, Samuel

    2008-03-01

    Forces exerted by and on adherent cells are important for many physiological processes such as wound healing and tissue formation. In addition, recent experiments have shown that stem cell differentiation is controlled, at least in part, by the elasticity of the surrounding matrix. Using a simple theoretical model that includes the forces due to both the mechanosensitive nature of cells and the elastic response of the matrix, we predict the dynamics of orientation of cells. The model predicts many features observed in measurements of cellular forces and orientation including the increase with time of the forces generated by cells in the absence of applied stress and the consequent decrease of the force in the presence of quasi-static stresses. We also explain the puzzling observation of parallel alignment of cells for static and quasi-static stresses and of nearly perpendicular alignment for dynamically varying stresses. In addition, we predict the response of the cellular orientation to a sinusoidally varying applied stress as a function of frequency. The dependence of the cell orientation angle on the Poisson ratio of the surrounding material can be used to distinguish systems in which cell activity is controlled by stress from those where cell activity is controlled by strain. Reference: Nature Physics, vol. 3, pp 655 (2007).

  13. 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

  14. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    PubMed

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  15. Thyroid Hormone Response Element Half-Site Organization and Its Effect on Thyroid Hormone Mediated Transcription

    PubMed Central

    Paquette, Martin A.; Atlas, Ella; Wade, Mike G.; Yauk, Carole L.

    2014-01-01

    Thyroid hormone (TH) exerts its effects by binding to the thyroid hormone receptor (TR), which binds to TH response elements (TREs) to regulate target gene expression. We investigated the relative ability of liganded homodimers TR and retinoid X receptor (RXR), and the heterodimer TR/RXR, to regulate gene expression for the TRE half-site organizations: direct repeat 4 (DR4), inverted repeat 0 (IR0) and everted repeat 6 (ER6). Luciferase reporter assays using a DR4 TRE suggest that both the TR homodimer and TR/RXR heterodimer regulate luciferase expression in the presence of their respective ligands. However, in the presence of the IR0 TRE, transfection with TR/RXR and RXR alone increased luciferase activity and there was no effect of TR alone. The presence of 9-cis-retinoic acid was necessary for luciferase expression, whereas TH treatment alone was insufficient. For the ER6 TRE, transfection with TR/RXR, TR alone and RXR alone (in the presence of their respective ligands) all caused a significant increase in luciferase activity. When both ligands were present, transfection with both TR/RXR caused more activation. Finally, we investigated the efficacy of the TR-antagonist 1–850 in inhibiting transcription by TR or TR/RXR at DR4 and ER6 TREs. We found that 1–850 did not suppress luciferase activation in the presence of TR/RXR for the ER6 TRE, suggesting conformational changes of the ligand binding domain of the TR when bound to different TRE half-site organizations. Collectively, the findings indicate that there are fundamental differences between TRE configurations that affect nuclear receptor interactions with the response element and ability to bind ligands and antagonists. PMID:24971931

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

    PubMed Central

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

    2015-01-01

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

  17. Evaluation of noise level and site response at Mt. Etna volcano and Aeolian Islands

    NASA Astrophysics Data System (ADS)

    D Amico, S.; Giampiccolo, E.; Maiolino, V.; Patanè, D.; Ursino, A.

    2003-04-01

    The aim of this work was to test the quality of the sites where the stations of the INGV-CT seismic network are installed. This because most of the installations will be soon improved with new broad-band sensors, which require a low level of background noise. Therefore, we investigated the noise level and estimated the site response at the seismic stations deployed at Mt. Etna and at Aeolian Islands, in order to evidence possible disturbs which can be related to anthropic activity, environmental factors and/or to the local soil conditions. Noise measurements were carried out using a portable digital seismic station equipped with a 3-component, 20 s sensor. The acquisition was performed both inside the vault structures where the remote stations are located and in proximity of them, on the outcropping terrain. The noise spectra were compared with the NLNM (New Low Noise Model) and NHNM (New High Noise Model) models described by Peterson (1993). A preliminary estimate of site response at each station, by applying the Nakamura (1989) technique, was also performed. The obtained results show, for some stations, higher noise levels mainly due to volcanic tremor and/or bad soil conditions. Moreover, in several cases, vault design need to be deeply reviewed and for some installations the substitution of the sites is required. References Nakamura, Y., (1989). A method for dynamic characteristics estimation of subsurface using microtremor on the ground surface. Quarterly R of Report RTRI, 30, 25-33. Peterson, J., (1993). Observations and modelling of background seismic noise. Open File Report 93-322, U. S. Geological Survey, Albuquerque, NM.

  18. Evaluation of criticality alarm response at the WEMCO Fernald site

    SciTech Connect

    Broadhead, B.L.; Childs, R.L.; Westfall, R.M.; Parks, C.V.

    1992-11-01

    This work quantifies the expected dose rates at a series of criticality alarm locations due to several postulated criticality accidents at the Westinghouse Environmental MANAGEMENT COMPANY OF OHIO (WEMCO) Fernald site. One- and two-dimensional discrete- ordinates calculations were performed for seven different shielding configurations using leakage spectra corresponding to two specific postulated critical events. In addition, an estimate of the gaseous fission products released during the hypothetical accident was made using ORIGEN-S point-depletion code.

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

    PubMed

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

    2016-01-01

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

  20. 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.

  1. 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

  2. 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

  3. The Copper Active Site of CBM33 Polysaccharide Oxygenases

    PubMed Central

    2013-01-01

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

  4. A kinetic description for sodium and potassium effects on (Na+ plus K+)-adenosine triphosphatase: a model for a two-nonequivalent site potassium activation and an analysis of multiequivalent site models for sodium activation.

    PubMed

    Lindenmayer, G E; Schwartz, A; Thompson, H K

    1974-01-01

    1. Dissociation constants for sodium and potassium of a site that modulates the rate of ouabain-(Na(+)+K(+))-ATPase interaction were applied to models for potassium activation of (Na(+)+K(+))-ATPase. The constants for potassium (0.213 mM) and for sodium (13.7 mM) were defined, respectively, as activation constant, K(a) and inhibitory constant, K(i).2. Tests of the one- and the two-equivalent site models, that describe sodium and potassium competition, revealed that neither model adequately predicts the activation effects of potassium in the presence of 100 or 200 mM sodium.3. The potassium-activation data, obtained at low potassium and high sodium, were explained by a two-nonequivalent site model where the dissociation constants of the first site are 0.213 mM for potassium and 13.7 mM for sodium. The second site was characterized by dissociation constants of 0.091 mM for potassium and 74.1 mM for sodium.4. The two-nonequivalent site model adequately predicted the responses to concentrations of potassium between 0.25 and 5 mM in the presence of 100-500 mM sodium. At lower sodium concentrations the predicted responses formed an upper limit for the function of observed activities. This limit was reached at lower concentrations of potassium and higher concentrations of sodium, which inferred saturation of the sodium-activation sites with sodium.5. Sodium-activation data were corrected for sodium interaction with potassium-activation sites by use of the two-nonequivalent site model for potassium activation. Tests of equivalent site models suggested that the corrected data for sodium activation may be most consistent with a model that has three-equivalent sites. Other multiequivalent site models (n = 2, 4, 5 or 6), however, cannot be statistically eliminated as possibilities. The three-equivalent site activation model was characterized by dissociation constants of 1.39 mM for sodium and 11.7 mM for potassium. The system theoretically would be half-maximally activated by

  5. 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.

  6. Investigations in site response from ground motion observations in vertical arrays

    NASA Astrophysics Data System (ADS)

    Baise, Laurie Gaskins

    The aim of the research is to improve the understanding of earthquake site response and to improve the techniques available to investigate issues in this field. Vertical array ground motion data paired with the empirical transfer function (ETF) methodology is shown to accurately characterize site response. This manuscript draws on methods developed in the field of signal processing and statistical time series analysis to parameterize the ETF as an autoregressive moving-average (ARMA) system which is justified theoretically, historically, and by example. Site response is evaluated at six sites in California, Japan, and Taiwan using ETF estimates, correlation analysis, and full waveform modeling. Correlation analysis is proposed as a required data quality evaluation imperative to any subsequent site response analysis. ETF estimates and waveform modeling are used to decipher the site response at sites with simple and complex geologic structure, which provide simple time-invariant and time-variant methods for evaluating both linear site transfer functions and nonlinear site response for sites experiencing liquefaction of the soils. The Treasure and Yerba Buena Island sites, however, require 2-D waveform modeling to accurately evaluate the effects of the shallow sedimentary basin. ETFs are used to characterize the Port Island site and corresponding shake table tests before, during, and after liquefaction. ETFs derived from the shake table tests were demonstrated to consistently predict the linear field ground response below 16 m depth and the liquefied behavior above 15 m depth. The liquefied interval response was demonstrated to gradually return to pre-liquefied conditions within several weeks of the 1995 Hyogo-ken Nanbu earthquake. Both the site's and the shake table test's response were shown to be effectively linear up to 0.5 g in the native materials below 16 m depth. The effective linearity of the site response at GVDA, Chiba, and Lotting up to 0.1 g, 0.33 g, and

  7. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  8. Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

    PubMed Central

    Zorrilla de San Martin, Javier; Jalil, Abdelali

    2015-01-01

    Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABAARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABAA autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca2+ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl−]i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABAA channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Nav-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABAA autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity. PMID:26621773

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

    PubMed Central

    Elias, Mikael; Chabriere, Eric

    2013-01-01

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

  10. 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

  11. 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.

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

    SciTech Connect

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

    1986-05-01

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

  13. 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

  14. 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.

  15. Site Response in Las Vegas Valley, Nevada from NTS Explosions and Earthquake Data

    NASA Astrophysics Data System (ADS)

    Rodgers, Arthur; Tkalcic, Hrvoje; McCallen, David; Larsen, Shawn; Snelson, Catherine

    2006-01-01

    We report site response in Las Vegas Valley (LVV) from historical recordings of Nevada Test Site (NTS) nuclear explosions and earthquake recordings from permanent and temporary seismic stations. Our data set significantly improves the spatial coverage of LVV over previous studies, especially in the northern, deeper parts of the basin. Site response at stations in LVV was measured for frequencies in the range 0.2 5.0 Hz using Standard Spectral Ratios (SSR) and Horizontal-Vertical Spectral Ratios (HVR). For the SSR measurements we used a reference site (approximately NEHRP B ``rock'' classification) located on Frenchman Mountain outside the basin. Site response at sedimentary sites is variable in LVV with average amplifications approaching a factor of 10 at some frequencies. We observed peaks in the site response curves at frequencies clustered near 0.6, 1.2 and 2.0 Hz, with some sites showing additional lower amplitude peaks at higher frequencies. The spatial pattern of site response is strongly correlated with the reported depth to basement for frequencies between 0.2 and 3.0 Hz, although the frequency of peak amplification does not show a similar correlation. For a few sites where we have geotechnical shear velocities, the amplification shows a correlation with the average upper 30-meter shear velocities, V 30. We performed two-dimensional finite difference simulations and reproduced the observed peak site amplifications at 0.6 and 1.2 Hz with a low velocity near-surface layer with shear velocities 600 750 m/s and a thickness of 100 200 m. These modeling results indicate that the amplitude and frequencies of site response peaks in LVV are strongly controlled by shallow velocity structure.

  16. Stress-induced enhancement of leukocyte trafficking into sites of surgery or immune activation

    NASA Astrophysics Data System (ADS)

    Viswanathan, Kavitha; Dhabhar, Firdaus S.

    2005-04-01

    Effective immunoprotection requires rapid recruitment of leukocytes into sites of surgery, wounding, infection, or vaccination. In contrast to immunosuppressive chronic stressors, short-term acute stressors have immunoenhancing effects. Here, we quantify leukocyte infiltration within a surgical sponge to elucidate the kinetics, magnitude, subpopulation, and chemoattractant specificity of an acute stress-induced increase in leukocyte trafficking to a site of immune activation. Mice acutely stressed before sponge implantation showed 200-300% higher neutrophil, macrophage, natural killer cell, and T cell infiltration than did nonstressed animals. We also quantified the effects of acute stress on lymphotactin- (LTN; a predominantly lymphocyte-specific chemokine), and TNF-- (a proinflammatory cytokine) stimulated leukocyte infiltration. An additional stress-induced increase in infiltration was observed for neutrophils, in response to TNF-, macrophages, in response to TNF- and LTN, and natural killer cells and T cells in response to LTN. These results show that acute stress initially increases trafficking of all major leukocyte subpopulations to a site of immune activation. Tissue damage-, antigen-, or pathogen-driven chemoattractants subsequently determine which subpopulations are recruited more vigorously. Such stress-induced increases in leukocyte trafficking may enhance immunoprotection during surgery, vaccination, or infection, but may also exacerbate immunopathology during inflammatory (cardiovascular disease or gingivitis) or autoimmune (psoriasis, arthritis, or multiple sclerosis) diseases. chemokine | psychophysiological stress | surgical sponge | wound healing | lymphotactin

  17. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    PubMed

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase. PMID:17850513

  18. 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.

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

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

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

  20. Trypanosoma cruzi Entrance through Systemic or Mucosal Infection Sites Differentially Modulates Regional Immune Response Following Acute Infection in Mice

    PubMed Central

    de Meis, Juliana; Barreto de Albuquerque, Juliana; Silva dos Santos, Danielle; Farias-de-Oliveira, Désio Aurélio; Berbert, Luiz Ricardo; Cotta-de-Almeida, Vinícius; Savino, Wilson

    2013-01-01

    Acute Chagas disease is characterized by a systemic infection that leads to the strong activation of the adaptive immune response. Outbreaks of oral contamination by the infective protozoan Trypanosoma cruzi are frequent in Brazil and other Latin American countries, and an increased severity of clinical manifestations and mortality is observed in infected patients. These findings have elicited questions about the specific responses triggered after T. cruzi entry via mucosal sites, possibly modulating local immune mechanisms, and further impacting regional and systemic immunity. Here, we provide evidence for the existence of differential lymphoid organ responses in experimental models of acute T. cruzi infection. PMID:23898334

  1. 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

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

    PubMed

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

    2015-05-01

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

  3. 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

  4. Site characterization and site response in Port-au-Prince, Haiti

    USGS Publications Warehouse

    Hough, Susan E.; Yong, Alan K.; Altidor, Jean Robert; Anglade, Dieuseul; Given, Douglas D.; Mildor, Saint-Louis

    2011-01-01

    Waveform analysis of aftershocks of the Mw7.0 Haiti earthquake of 12 January 2010 reveals amplification of ground motions at sites within the Cul de Sac valley in which Port-au-Prince is situated. Relative to ground motions recorded at a hard-rock reference site, peak acceleration values are amplified by a factor of approximately 1.8 at sites on low-lying Mio-Pliocene deposits in central Port-au-Prince and by a factor of approximately 2.5–3 on a steep foothill ridge in the southern Port-au-Prince metropolitan region. The observed amplitude, predominant periods, variability, and polarization of amplification are consistent with predicted topographic amplification by a steep, narrow ridge. A swath of unusually high damage in this region corresponds with the extent of the ridge where high weak-motion amplifications are observed. We use ASTER (Advanced Spaceborne Thermal Emission and Reflection Radiometer) imagery to map local geomorphology, including characterization of both near-surface and of small-scale topographic structures that correspond to zones of inferred amplification.

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

    PubMed

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

    2016-01-01

    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

  6. Site-specific cleavage of the transactivation response site of human immunodeficiency virus RNA with a tat-based chemical nuclease.

    PubMed Central

    Jayasena, S D; Johnston, B H

    1992-01-01

    tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo. Images PMID:1565648

  7. Classical Nuclear Hormone Receptor Activity as a Mediator of Complex Concentration Response Relationships for Endocrine Active Compounds

    PubMed Central

    Cookman, Clifford J.; Belcher, Scott M.

    2014-01-01

    Nonmonotonic concentration response relationships are frequently observed for endocrine active ligands that act via nuclear receptors. The curve of best fit for nonmonotonic concentration response relationships are often inverted U-shaped with effects at intermediate concentrations that are different from effects at higher or lower concentrations. Cytotoxicity is a major mode of action responsible for inverted U-shaped concentration response relationships. However, evidence suggests that ligand selectivity, activation of multiple molecular targets, concerted regulation of multiple opposing endpoints, and multiple ligand binding sites within nuclear receptors also contribute to nonmonotonic concentration response relationships of endocrine active ligands. This review reports the current understanding of mechanisms involved in classical nuclear receptor mediated nonmonotonic concentration response relationships with a focus on studies published between 2012 and 2014. PMID:25299165

  8. Functional activity of the porcine Gnrhr2 gene promoter in testis-derived cells is partially conferred by nuclear factor-κB, specificity protein 1 and 3 (SP1/3) and overlapping early growth response 1/SP1/3 binding sites.

    PubMed

    Brauer, Vanessa M; Wiarda-Bell, Jocelyn R; Desaulniers, Amy T; Cederberg, Rebecca A; White, Brett R

    2016-08-10

    Unlike the classical gonadotropin-releasing hormone (GnRH1), the second mammalian isoform (GnRH2) is ubiquitously expressed, suggesting a divergent function. Indeed, we demonstrated that GnRH2 governs LH-independent testosterone secretion in porcine testes via interaction with its receptor (GnRHR2) on Leydig cells. Transient transfections with luciferase reporter vectors containing 3009bp of 5' flanking sequence for the porcine Gnrhr2 gene (-3009pGL3) revealed promoter activity in all 15 cell lines examined, including swine testis-derived (ST) cells. Therefore, ST cells were utilized to explore the molecular mechanisms underlying transcriptional regulation of the porcine Gnrhr2 gene in the testis. Reporter plasmids containing progressive 5' deletions of the Gnrhr2 promoter indicated that the -708/-490 region contained elements critical to promoter activity. Electrophoretic mobility shift assays (EMSAs) with radiolabeled oligonucleotides spanning the -708/-490bp region and ST nuclear extracts, identified specific binding complexes for the -513/-490, -591/-571 and -606/-581bp segments of promoter. Antibody addition to EMSAs indicated that the p65 and p52 subunits of nuclear factor-κB (NF-κB) comprised the specific complex bound to the oligonucleotide probe for the -513/-490bp promoter region, specificity protein (SP) 1 and 3 bound the -591/-571bp probe and early growth response 1 (EGR1), SP1 and SP3 bound the -606/-581 radiolabeled oligonucleotide. Transient transfections with vectors containing mutations of the NF-κB (-499/-493), SP1/3 (-582/-575) or overlapping EGR1/SP1/3 (-597/-587) binding sites reduced luciferase activity by 26%, 61% and 56%, respectively (P<0.05). Thus, NF-κB, SP1/3 and overlapping EGR1/SP1/3 binding sites are critical to expression of the porcine Gnrhr2 gene in ST cells. PMID:27134031

  9. 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

  10. Identification of a novel interleukin-6 response element containing an Ets-binding site and a CRE-like site in the junB promoter.

    PubMed Central

    Nakajima, K; Kusafuka, T; Takeda, T; Fujitani, Y; Nakae, K; Hirano, T

    1993-01-01

    Interleukin-6 (IL-6) activation of the immediate-early gene junB has been shown to require both a tyrosine kinase and an unknown 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7)-sensitive pathway. Here we report the identification and characterization of an IL-6 immediate-early response element in the junB promoter (designated JRE-IL6) in HepG2 cells. The JRE-IL6 element, located at -149 to -124, contains two DNA motifs, an Ets-binding site (EBS) (CAGGAAGC) and a CRE-like site (TGACGCGA). Functional studies using variously mutated JRE-IL6 elements showed that both motifs were necessary and sufficient for IL-6 response of the promoter. The EBS of the JRE-IL6 element (JEBS) appears to bind a protein in the Ets family or a related protein which could also form a major complex with the EBSs of the murine sarcoma virus long terminal repeat or human T-cell leukemia virus type 1 long terminal repeat. The CRE-like site appears to weakly bind multiple CREB-ATF family proteins. Despite the similarity in the structure between the JRE-IL6 element and the polyomavirus enhancer PyPEA3, composed of an EBS and an AP1-binding site and known to be activated by a variety of oncogene signals, JRE-IL6 could not be activated by activated Ha-Ras, Raf-1, or 12-O-tetradecanoylphorbol-13-acetate. We show that IL-6 activates JRE-IL6 through an H7-sensitive pathway that does not involve protein kinase C, cyclic AMP-dependent kinase, Ca(2+)- or calmodulin-dependent kinases, Ras, Raf-1, or NF-IL6 (C/EBP beta). The combination of JEBS and the CRE-like site appears to form the basis for the selective and efficient response of JRE-IL6 to IL-6 signals, but not to signals generated by activated Ha-Ras, Raf-1, or protein kinase C. Images PMID:8386318

  11. Wave propagation and site response in the Santa Clara Valley

    USGS Publications Warehouse

    Fletcher, Joe B.; Boatwright, J.; Lindh, A.G.

    2003-01-01

    Forty-two portable digital instruments were deployed across the Santa Clara Valley from June until early November 1998; this array recorded 14 small and moderate local events and 7 large teleseismic events. We analyze the ground motion from these events to determine station delays and relative site amplification within the Valley. P waves from an event at the southern edge of the valley are early (??t > -0.35 sec) at stations over an axial ridge in the basement interface in the middle of the valley, but late (??t < 0.20 sec) for stations over the Cupertino and Evergreen basins to either side. The S-wave delays are approximately twice as large. Teleseismic P-waves from an M = 7.0 event beneath the Bonin Islands show a similar pattern in travel-time delays. The P waves are amplified by factors of 1.5-3 for frequencies below 2 Hz at stations within either basin, compared with stations on the axial ridge. The P-wave coda appear enhanced at 2-3 sec, but coda Q estimates at frequencies from 0.2 to 1.1 Hz are not markedly different at stations over the basin compared with stations on the ridge with the possible exceptions of consistently high values over the northern end of the Evergreen Basin. We invert the S-wave spectra for site-specific attenuation and amplification from the 14 local events by assuming a common source spectra for each event, 1/r geometrical spreading, and constraining the inversion using the 30-m velocity profile at four stations in the array. The largest amplifications occurred in the 1- to 6-Hz band at stations near the northwest edge of the Evergreen basin. While the highest amplifications occur at stations with the lowest S-wave velocities, the scatter obscures the correlation between velocity and amplification. The stations in the basins are characterized by higher attenuation than the stations on the basement ridge.

  12. Hydrologic response and radionuclide transport following fire at semiarid sites.

    PubMed

    Johansen, M P; Hakonson, T E; Whicker, F W; Simanton, J R; Stone, J J

    2001-01-01

    Infrequent, high-impact events such as wildfires, droughts, biological shifts, floods, and mechanical disturbances can greatly change land surfaces, including vegetative cover and soil characteristics, which in turn can trigger high rates of hydrologic erosion and associated transport of sediments and sediment-sorbed contaminants. Where persistent soil contamination exists, infrequent mobilization of contaminants may dominate in determining long-term risks to human and ecological receptors. Among these infrequent events, fire stands out as having the capacity to cause large increases in sediment transport. This study measured runoff, sediment yield, and mobility of sediment-sorbed contamination (137Cs) on burned and unburned plots at the Waste Isolation Pilot Plant, New Mexico (WIPP), and the Rocky Flats Environmental Technology Site, Colorado (RFETS). Results showed that 137Cs transport from burned plots was up to 22 times greater than that from unburned plots at WIPP and 4 times greater at RFETS. Associated runoff was up to 12 times greater on burned plots at WIPP and sediment yields up to 6 times greater. Further, 137Cs concentrations in transported sediments were enriched compared with parent soils (expressed as enrichment ratio) by a factor of 2.3 at WIPP, and 1.3 at RFETS. However, enrichment ratios were not significantly different in sediments from burned and unburned plots. Our results provide new data on the effects of fire on the transport of sediment-sorbed contaminants, and demonstrate that rare events such as fire can greatly increase contaminant mobility. PMID:11790008

  13. 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

  14. Quantifying Fast and Slow Responses of Terrestrial Carbon Exchange across a Water Availability Gradient in North American Flux Sites

    NASA Astrophysics Data System (ADS)

    Biederman, J. A.; Scott, R. L.; Goulden, M.

    2014-12-01

    Climate change is predicted to increase the frequency and severity of water limitation, altering terrestrial ecosystems and their carbon exchange with the atmosphere. Here we compare site-level temporal sensitivity of annual carbon fluxes to interannual variations in water availability against cross-site spatial patterns over a network of 19 eddy covariance flux sites. This network represents one order of magnitude in mean annual productivity and includes western North American desert shrublands and grasslands, savannahs, woodlands, and forests with continuous records of 4 to 12 years. Our analysis reveals site-specific patterns not identifiable in prior syntheses that pooled sites. We interpret temporal variability as an indicator of ecosystem response to annual water availability due to fast-changing factors such as leaf stomatal response and microbial activity, while cross-site spatial patterns are used to infer ecosystem adjustment to climatic water availability through slow-changing factors such as plant community and organic carbon pools. Using variance decomposition, we directly quantify how terrestrial carbon balance depends on slow- and fast-changing components of gross ecosystem production (GEP) and total ecosystem respiration (TER). Slow factors explain the majority of variance in annual net ecosystem production (NEP) across the dataset, and their relative importance is greater at wetter, forest sites than desert ecosystems. Site-specific offsets from spatial patterns of GEP and TER explain one third of NEP variance, likely due to slow-changing factors not directly linked to water, such as disturbance. TER and GEP are correlated across sites as previously shown, but our site-level analysis reveals surprisingly consistent linear relationships between these fluxes in deserts and savannahs, indicating fast coupling of TER and GEP in more arid ecosystems. Based on the uncertainty associated with slow and fast factors, we suggest a framework for improved

  15. 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

  16. 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.

  17. The Histamine N-Methyltransferase T105I Polymorphism Affects Active Site Structure and Dynamics†

    PubMed Central

    Rutherford, Karen; Parson, William W.; Daggett, Valerie

    2010-01-01

    Histamine N-methyltransferase (HNMT) is the sole enzyme responsible for inactivating histamine in the mammalian brain. The human HNMT gene contains a common threonine-isoleucine polymorphism at residue 105, distal from the active site. The 105I variant has decreased activity and lower protein levels relative to the 105T protein. Crystal structures of both variants have been solved, but reveal little regarding how the T105I polymorphism affects activity. We performed molecular dynamics simulations of both 105T and 105I at 37°C to explore the structural and dynamic consequences of the polymorphism. The simulations indicate that replacing Thr with the larger Ile residue leads to greater burial of residue 105 and heightened packing interactions between residue105 and residues within helix α3 and strand β3. This altered packing is directly translated to the active site resulting in the reorientation of several co-substrate-binding residues. The simulations also show that the hydrophobic histamine-binding domain in both proteins undergoes a large-scale breathing motion that exposes key catalytic residues and lessens the hydrophobicity of the substrate-binding site. PMID:18154359

  18. 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

  19. Y-12 site-specific earthquake response analysis and soil liquefaction assessment

    SciTech Connect

    Ahmed, S.B.; Hunt, R.J.; Manrod, W.E. III

    1995-09-29

    A site-specific earthquake response analysis and soil liquefaction assessment were performed for the Oak Ridge Y-12 Plant. The main purpose of these studies was to use the results of the analyses for evaluating the safety of the performance category -1, -2, and -3 facilities against the natural phenomena seismic hazards. Earthquake response was determined for seven (7), one dimensional soil columns (Fig. 12) using two horizontal components of the PC-3 design basis 2000-year seismic event. The computer program SHAKE 91 (Ref. 7) was used to calculate the absolute response accelerations on top of ground (soil/weathered shale) and rock outcrop. The SHAKE program has been validated for horizontal response calculations at periods less than 2.0 second at several sites and consequently is widely accepted in the geotechnical earthquake engineering area for site response analysis.

  20. 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

  1. 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

  2. ANL site response for the DOE FY1994 information resources management long-range plan

    SciTech Connect

    Boxberger, L.M.

    1992-03-01

    Argonne National Laboratory's ANL Site Response for the DOE FY1994 Information Resources Management (IRM) Long-Range Plan (ANL/TM 500) is one of many contributions to the DOE information resources management long-range planning process and, as such, is an integral part of the DOE policy and program planning system. The Laboratory has constructed this response according to instructions in a Call issued in September 1991 by the DOE Office of IRM Policy, Plans and Oversight. As one of a continuing series, this Site Response is an update and extension of the Laboratory's previous submissions. The response contains both narrative and tabular material. It covers an eight-year period consisting of the base year (FY1991), the current year (FY1992), the budget year (FY1993), the plan year (FY1994), and the out years (FY1995-FY1998). This Site Response was compiled by Argonne National Laboratory's Computing and Telecommunications Division (CTD), which has the responsibility to provide leadership in optimizing computing and information services and disseminating computer-related technologies throughout the Laboratory. The Site Response consists of 5 parts: (1) a site overview, describes the ANL mission, overall organization structure, the strategic approach to meet information resource needs, the planning process, major issues and points of contact. (2) a software plan for DOE contractors, Part 2B, Software Plan FMS plan for DOE organizations, (3) computing resources telecommunications, (4) telecommunications, (5) printing and publishing.

  3. ANL site response for the DOE FY1994 information resources management long-range plan

    SciTech Connect

    Boxberger, L.M.

    1992-03-01

    Argonne National Laboratory`s ANL Site Response for the DOE FY1994 Information Resources Management (IRM) Long-Range Plan (ANL/TM 500) is one of many contributions to the DOE information resources management long-range planning process and, as such, is an integral part of the DOE policy and program planning system. The Laboratory has constructed this response according to instructions in a Call issued in September 1991 by the DOE Office of IRM Policy, Plans and Oversight. As one of a continuing series, this Site Response is an update and extension of the Laboratory`s previous submissions. The response contains both narrative and tabular material. It covers an eight-year period consisting of the base year (FY1991), the current year (FY1992), the budget year (FY1993), the plan year (FY1994), and the out years (FY1995-FY1998). This Site Response was compiled by Argonne National Laboratory`s Computing and Telecommunications Division (CTD), which has the responsibility to provide leadership in optimizing computing and information services and disseminating computer-related technologies throughout the Laboratory. The Site Response consists of 5 parts: (1) a site overview, describes the ANL mission, overall organization structure, the strategic approach to meet information resource needs, the planning process, major issues and points of contact. (2) a software plan for DOE contractors, Part 2B, ``Software Plan FMS plan for DOE organizations, (3) computing resources telecommunications, (4) telecommunications, (5) printing and publishing.

  4. Study case - Induced Polarization response from a BTEX contaminated site in Brazil

    NASA Astrophysics Data System (ADS)

    Ustra, A.; Elis, V.; Minozzo, M.

    2011-12-01

    A hydrocarbon contaminated site in Brazil was investigated using DC-resistivity and Induced Polarization (IP) methods. The study area is a chemical industry facility that manufactures paint for automobiles. The industrial process involves the use of many hydrocarbon derivative products, including BTEX (benzene, toluene, ethyl benzene and xylene) and organic chlorides. The area was contaminated by some (not documented) accidental spills of BTEX throughout many years. Monitoring wells revealed concentrations from a few ppm to hundreds ppm of BTEX around the area, as well as other compounds. Two soil samples were collected from an area where some spills where known to have happened. Soil analyses of these samples found the presence of microbes, and therefore biodegradation is believed to be occurring at the site. The objective of this study is to relate the IP response distribution to the presence of contamination and/or microbial activity. The geophysical survey consisted in a rectangular mesh composed of 15 parallel lines with 60 meters of extension, using dipole-dipole array. Lines were spaced by 3 meters. Metallic electrodes were used for current injection, and non-polarizing electrodes (Cu/CuSO4) for potential measurement. Current was injected in cycles of 2 seconds. IP measurements were recorded after 160 milliseconds delay of current shut off, and integration time windows were 120, 220, 420, and 820 milliseconds. All data were concatenated into a single data set and submitted to 3D inversion routine. A conductive zone (resistivity less than 100 ohm.m and chargeability less than 2mV/V) was observed where microbes were found. This feature was interpreted as possibly due to natural biodegradation process, that increases total dissolved salts as a result of mineral weathering by organic acids produced in the degradation process. Normalized chargeability (chargeability divided by resistivity) showed an enhanced polarization zone where microbes were detected. This

  5. Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

    PubMed Central

    Lee, Minho; Shin, Eunkyoung; Jeon, Che Ok; Cha, Chang-Jun; Han, Seung Hyun; Kim, Su-Jin; Lee, Sang-Won; Lee, Younghoon; Ha, Nam-Chul

    2011-01-01

    Escherichia coli RNase E contains a site that selectively binds to RNAs containing 5′-monophosphate termini, increasing the efficiency of endonucleolytic cleavage of these RNAs. Random mutagenesis of N-Rne, the N-terminal catalytic region of RNase E, identified a hyperactive variant that remains preferentially responsive to phosphorylation at 5′ termini. Biochemical analyses showed that the mutation (Q36R), which replaces glutamine with arginine at a position distant from the catalytic site, increases formation of stable RNA-protein complexes without detectably affecting the enzyme's secondary or tertiary structure. Studies of cleavage of fluorogenic substrate and EMSA experiments indicated that the Q36R mutation increases catalytic activity and RNA binding. however, UV crosslinking and mass spectrometry studies suggested that the mutant enzyme lacks an RNA binding site present in its wild-type counterpart. Two substrate-bound tryptic peptides, 65HGFLPLK71—which includes amino acids previously implicated in substrate binding and catalysis—and 24LYDLDIESPGHEQK37—which includes the Q36 locus—were identified in wild-type enzyme complexes, whereas only the shorter peptide was observed for complexes containing Q36R. Our results identify a novel RNase E locus that disparately affects the number of substrate binding sites and catalytic activity of the enzyme. We propose a model that may account for these surprising effects. PMID:22186084

  6. Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity.

    PubMed

    Barinka, Cyril; Sácha, Pavel; Sklenár, Jan; Man, Petr; Bezouska, Karel; Slusher, Barbara S; Konvalinka, Jan

    2004-06-01

    Glutamate carboxypeptidase II (GCPII) is a membrane peptidase expressed in the prostate, central and peripheral nervous system, kidney, small intestine, and tumor-associated neovasculature. The GCPII form expressed in the central nervous system, termed NAALADase, is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG) yielding free glutamate in the synaptic cleft, and is implicated in various pathologic conditions associated with glutamate excitotoxicity. The prostate form of GCPII, termed prostate-specific membrane antigen (PSMA), is up-regulated in cancer and used as an effective prostate cancer marker. Little is known about the structure of this important pharmaceutical target. As a type II membrane protein, GCPII is heavily glycosylated. In this paper we show that N-glycosylation is vital for proper folding and subsequent secretion of human GCPII. Analysis of the predicted N-glycosylation sites also provides evidence that these sites are critical for GCPII carboxypeptidase activity. We confirm that all predicted N-glycosylation sites are occupied by an oligosaccharide moiety and show that glycosylation at sites distant from the putative catalytic domain is critical for the NAAG-hydrolyzing activity of GCPII calling the validity of previously described structural models of GCPII into question. PMID:15152093

  7. In Operando Identification of Geometrical-Site-Dependent Water Oxidation Activity of Spinel Co3O4.

    PubMed

    Wang, Hsin-Yi; Hung, Sung-Fu; Chen, Han-Yi; Chan, Ting-Shan; Chen, Hao Ming; Liu, Bin

    2016-01-13

    Spinel Co3O4, comprising two types of cobalt ions: one Co(2+) in the tetrahedral site (Co(2+)(Td)) and the other two Co(3+) in the octahedral site (Co(3+)(Oh)), has been widely explored as a promising oxygen evolution reaction (OER) catalyst for water electrolysis. However, the roles of two geometrical cobalt ions toward the OER have remained elusive. Here, we investigated the geometrical-site-dependent OER activity of Co3O4 catalyst by substituting Co(2+)(Td) and Co(3+)(Oh) with inactive Zn(2+) and Al(3+), respectively. Following a thorough in operando analysis by electrochemical impedance spectroscopy and X-ray absorption spectroscopy, it was revealed that Co(2+)Td site is responsible for the formation of cobalt oxyhydroxide (CoOOH), which acted as the active site for water oxidation. PMID:26710084

  8. Semiparametric Geographically Weighted Response Curves with Application to Site Specific Agriculture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lack of basic knowledge about spatial and treatment varying crop response to irrigation hinders irrigation management and economic analysis for site-specific agriculture. One model that has been postulated for relating crop-specific economic quantities to irrigation is a quadratic response curve of...

  9. Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation.

    PubMed

    Beke-Somfai, Tamás; Lincoln, Per; Nordén, Bengt

    2013-02-01

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate. PMID:23345443

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

    PubMed

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

    2016-08-01

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

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

    PubMed

    Li, Chi-Hui; Tu, Shiao-Chun

    2005-10-01

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

  12. 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.

  13. The active site architecture in peroxiredoxins: a case study on Mycobacterium tuberculosis AhpE.

    PubMed

    Pedre, Brandán; van Bergen, Laura A H; Palló, Anna; Rosado, Leonardo A; Dufe, Veronica Tamu; Molle, Inge Van; Wahni, Khadija; Erdogan, Huriye; Alonso, Mercedes; Proft, Frank De; Messens, Joris

    2016-08-11

    Peroxiredoxins catalyze the reduction of peroxides, a process of vital importance to survive oxidative stress. A nucleophilic cysteine, also known as the peroxidatic cysteine, is responsible for this catalytic process. We used the Mycobacterium tuberculosis alkyl hydroperoxide reductase E (MtAhpE) as a model to investigate the effect of the chemical environment on the specificity of the reaction. Using an integrative structural (R116A - PDB ; F37H - PDB ), kinetic and computational approach, we explain the mutational effects of key residues in its environment. This study shows that the active site residues are specifically oriented to create an environment which selectively favours a reaction with peroxides. PMID:27471753

  14. HMGN proteins modulate chromatin regulatory sites and gene expression during activation of naïve B cells

    PubMed Central

    Zhang, Shaofei; Zhu, Iris; Deng, Tao; Furusawa, Takashi; Rochman, Mark; Vacchio, Melanie S.; Bosselut, Remy; Yamane, Arito; Casellas, Rafael; Landsman, David; Bustin, Michael

    2016-01-01

    The activation of naïve B lymphocyte involves rapid and major changes in chromatin organization and gene expression; however, the complete repertoire of nuclear factors affecting these genomic changes is not known. We report that HMGN proteins, which bind to nucleosomes and affect chromatin structure and function, co-localize with, and maintain the intensity of DNase I hypersensitive sites genome wide, in resting but not in activated B cells. Transcription analyses of resting and activated B cells from wild-type and Hmgn−/− mice, show that loss of HMGNs dampens the magnitude of the transcriptional response and alters the pattern of gene expression during the course of B-cell activation; defense response genes are most affected at the onset of activation. Our study provides insights into the biological function of the ubiquitous HMGN chromatin binding proteins and into epigenetic processes that affect the fidelity of the transcriptional response during the activation of B cell lymphocytes. PMID:27112571

  15. 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

  16. Modulating protein activity using tethered ligands with mutually exclusive binding sites

    PubMed Central

    Schena, Alberto; Griss, Rudolf; Johnsson, Kai

    2015-01-01

    The possibility to design proteins whose activities can be switched on and off by unrelated effector molecules would enable applications in various research areas, ranging from biosensing to synthetic biology. We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a ligand to the protein of interest results in an intramolecular ligand–protein interaction that can be disrupted through the presence of the effector. Specifically, we introduce a luciferase controlled by another protein, a human carbonic anhydrase whose activity can be controlled by proteins or small molecules in vitro and on living cells, and novel fluorescent and bioluminescent biosensors. PMID:26198003

  17. 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

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

    PubMed Central

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

    1982-01-01

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

  19. Characterization of active site residues of nitroalkane oxidase.

    PubMed

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

    2010-06-01

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

  20. 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.

  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. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. GITR Activation Positively Regulates Immune Responses against Toxoplasma gondii

    PubMed Central

    Costa, Frederico R. C.; Mota, Caroline M.; Santiago, Fernanda M.; Silva, Murilo V.; Ferreira, Marcela D.; Fonseca, Denise M.; Silva, João S.; Mineo, José R.; Mineo, Tiago W. P.

    2016-01-01

    Toxoplasma gondii is a widespread parasite responsible for causing clinical diseases especially in pregnant and immunosuppressed individuals. Glucocorticoid-induced TNF receptor (GITR), which is also known as TNFRS18 and belongs to the TNF receptor superfamily, is found to be expressed in various cell types of the immune system and provides an important costimulatory signal for T cells and myeloid cells. However, the precise role of this receptor in the context of T. gondii infection remains elusive. Therefore, the current study investigated the role of GITR activation in the immunoregulation mechanisms induced during the experimental infection of mice with T. gondii. Our data show that T. gondii infection slightly upregulates GITR expression in Treg cells and B cells, but the most robust increment in expression was observed in macrophages and dendritic cells. Interestingly, mice infected and treated with an agonistic antibody anti-GITR (DTA-1) presented a robust increase in pro-inflammatory cytokine production at preferential sites of parasite replication, which was associated with the decrease in latent brain parasitism of mice under treatment with DTA-1. Several in vivo and in vitro analysis were performed to identify the cellular mechanisms involved in GITR activation upon infection, however no clear alterations were detected in the phenotype/function of macrophages, Tregs and B cells under treatment with DTA-1. Therefore, GITR appears as a potential target for intervention during infection by the parasite Toxoplasma gondii, even though further studies are still necessary to better characterize the immune response triggered by GITR activation during T. gondii infection. PMID:27027302

  8. Analysis of ground response data at Lotung large-scale soil- structure interaction experiment site. Final report

    SciTech Connect

    Chang, C.Y.; Mok, C.M.; Power, M.S.

    1991-12-01

    The Electric Power Research Institute (EPRI), in cooperation with the Taiwan Power Company (TPC), constructed two models (1/4-scale and 1/2-scale) of a nuclear plant containment structure at a site in Lotung (Tang, 1987), a seismically active region in northeast Taiwan. The models were constructed to gather data for the evaluation and validation of soil-structure interaction (SSI) analysis methodologies. Extensive instrumentation was deployed to record both structural and ground responses at the site during earthquakes. The experiment is generally referred to as the Lotung Large-Scale Seismic Test (LSST). As part of the LSST, two downhole arrays were installed at the site to record ground motions at depths as well as at the ground surface. Structural response and ground response have been recorded for a number of earthquakes (i.e. a total of 18 earthquakes in the period of October 1985 through November 1986) at the LSST site since the completion of the installation of the downhole instruments in October 1985. These data include those from earthquakes having magnitudes ranging from M{sub L} 4.5 to M{sub L} 7.0 and epicentral distances range from 4.7 km to 77.7 km. Peak ground surface accelerations range from 0.03 g to 0.21 g for the horizontal component and from 0.01 g to 0.20 g for the vertical component. The objectives of the study were: (1) to obtain empirical data on variations of earthquake ground motion with depth; (2) to examine field evidence of nonlinear soil response due to earthquake shaking and to determine the degree of soil nonlinearity; (3) to assess the ability of ground response analysis techniques including techniques to approximate nonlinear soil response to estimate ground motions due to earthquake shaking; and (4) to analyze earth pressures recorded beneath the basemat and on the side wall of the 1/4 scale model structure during selected earthquakes.

  9. 76 FR 19766 - Agency Information Collection Activities OMB Responses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-08

    ... From the Federal Register Online via the Government Publishing Office ENVIRONMENTAL PROTECTION AGENCY Agency Information Collection Activities OMB Responses AGENCY: Environmental Protection Agency... 1808.06; Environmental Impact Assessment of Nongovernmental Activities in Antarctica (Renewal); 40...

  10. Spatial patterns of microbial diversity and activity in an aged creosote-contaminated site

    PubMed Central

    Mukherjee, Shinjini; Juottonen, Heli; Siivonen, Pauli; Lloret Quesada, Cosme; Tuomi, Pirjo; Pulkkinen, Pertti; Yrjälä, Kim

    2014-01-01

    Restoration of polluted sites via in situ bioremediation relies heavily on the indigenous microbes and their activities. Spatial heterogeneity of microbial populations, contaminants and soil chemical parameters on such sites is a major hurdle in optimizing and implementing an appropriate bioremediation regime. We performed a grid-based sampling of an aged creosote-contaminated site followed by geostatistical modelling to illustrate the spatial patterns of microbial diversity and activity and to relate these patterns to the distribution of pollutants. Spatial distribution of bacterial groups unveiled patterns of niche differentiation regulated by patchy distribution of pollutants and an east-to-west pH gradient at the studied site. Proteobacteria clearly dominated in the hot spots of creosote pollution, whereas the abundance of Actinobacteria, TM7 and Planctomycetes was considerably reduced from the hot spots. The pH preferences of proteobacterial groups dominating in pollution could be recognized by examining the order and family-level responses. Acidobacterial classes came across as generalists in hydrocarbon pollution whose spatial distribution seemed to be regulated solely by the pH gradient. Although the community evenness decreased in the heavily polluted zones, basal respiration and fluorescein diacetate hydrolysis rates were higher, indicating the adaptation of specific indigenous microbial populations to hydrocarbon pollution. Combining the information from the kriged maps of microbial and soil chemistry data provided a comprehensive understanding of the long-term impacts of creosote pollution on the subsurface microbial communities. This study also highlighted the prospect of interpreting taxa-specific spatial patterns and applying them as indicators or proxies for monitoring polluted sites. PMID:25105905

  11. Spatial patterns of microbial diversity and activity in an aged creosote-contaminated site.

    PubMed

    Mukherjee, Shinjini; Juottonen, Heli; Siivonen, Pauli; Lloret Quesada, Cosme; Tuomi, Pirjo; Pulkkinen, Pertti; Yrjälä, Kim

    2014-10-01

    Restoration of polluted sites via in situ bioremediation relies heavily on the indigenous microbes and their activities. Spatial heterogeneity of microbial populations, contaminants and soil chemical parameters on such sites is a major hurdle in optimizing and implementing an appropriate bioremediation regime. We performed a grid-based sampling of an aged creosote-contaminated site followed by geostatistical modelling to illustrate the spatial patterns of microbial diversity and activity and to relate these patterns to the distribution of pollutants. Spatial distribution of bacterial groups unveiled patterns of niche differentiation regulated by patchy distribution of pollutants and an east-to-west pH gradient at the studied site. Proteobacteria clearly dominated in the hot spots of creosote pollution, whereas the abundance of Actinobacteria, TM7 and Planctomycetes was considerably reduced from the hot spots. The pH preferences of proteobacterial groups dominating in pollution could be recognized by examining the order and family-level responses. Acidobacterial classes came across as generalists in hydrocarbon pollution whose spatial distribution seemed to be regulated solely by the pH gradient. Although the community evenness decreased in the heavily polluted zones, basal respiration and fluorescein diacetate hydrolysis rates were higher, indicating the adaptation of specific indigenous microbial populations to hydrocarbon pollution. Combining the information from the kriged maps of microbial and soil chemistry data provided a comprehensive understanding of the long-term impacts of creosote pollution on the subsurface microbial communities. This study also highlighted the prospect of interpreting taxa-specific spatial patterns and applying them as indicators or proxies for monitoring polluted sites. PMID:25105905

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

    PubMed

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

    2001-08-01

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

  13. 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.

  14. Dynamically Achieved Active Site Precision in Enzyme Catalysis

    PubMed Central

    2015-01-01

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

  15. Comment and response document for the final remedial action plan and site design for stabilization of the inactive uranium mill tailings sites at Slick Rock, Colorado. Revision 2

    SciTech Connect

    1996-05-01

    This document for the final remedial action plan and site design has been prepared for US Department of Energy Environmental Restoration Division as part of the Uranium Mill Tailings Remedial Action plan. Comments and responses are included for the site design for stabilization of the inactive uranium mill tailings sites at Slick Rock, Colorado.

  16. A case study of alternative site response explanatory variables in Parkfield, California

    USGS Publications Warehouse

    Thompson, E.M.; Baise, L.G.; Kayen, R.E.; Morgan, E.C.; Kaklamanos, J.

    2011-01-01

    The combination of densely-spaced strong-motion stations in Parkfield, California, and spectral analysis of surface waves (SASW) profiles provides an ideal dataset for assessing the accuracy of different site response explanatory variables. We judge accuracy in terms of spatial coverage and correlation with observations. The performance of the alternative models is period-dependent, but generally we observe that: (1) where a profile is available, the square-root-of-impedance method outperforms VS30 (average S-wave velocity to 30 m depth), and (2) where a profile is unavailable, the topographic-slope method outperforms surficial geology. The fundamental site frequency is a valuable site response explanatory variable, though less valuable than VS30. However, given the expense and difficulty of obtaining reliable estimates of VS30 and the relative ease with which the fundamental site frequency can be computed, the fundamental site frequency may prove to be a valuable site response explanatory variable for many applications. ?? 2011 ASCE.

  17. Protein Kinase A and Mitogen-Activated Protein Kinase Pathways Antagonistically Regulate Fission Yeast fbp1 Transcription by Employing Different Modes of Action at Two Upstream Activation Sites

    PubMed Central

    Neely, Lori A.; Hoffman, Charles S.

    2000-01-01

    A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions. To study this, we have investigated the transcriptional regulation of the Schizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study, we identified and characterized two cis-acting elements in the fbp1 promoter required for activation of fbp1 transcription. Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA. UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes. Transcriptional regulation of fbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We conclude that the PKA and MAPK signal transduction pathways regulate fbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site. PMID:10938120

  18. Mapping Site Response Parameters on Cal Poly Pomona Campus Using the Spectral Ratio Method

    NASA Astrophysics Data System (ADS)

    HO, K. Y. K.; Polet, J.

    2014-12-01

    Site characteristics are an important factor in earthquake hazard assessment. To better understand site response differences on a small scale, as well as the seismic hazard of the area, we develop site response parameter maps of Cal Poly Pomona campus. Cal Poly Pomona is located in southern California about 40 km east of Los Angeles, within 50 km of San Andreas Fault. The campus is situated on top of the San Jose Fault. With about twenty two thousand students on campus, it is important to know the site response in this area. To this end, we apply the Horizontal-to-Vertical (H/V) spectral ratio technique, which is an empirical method that can be used in an urban environment with no environmental impact. This well-established method is based on the computation of the ratio of vertical ambient noise ground motion over horizontal ambient noise ground motion as a function of frequency. By applying the spectral ratio method and the criteria from Site Effects Assessment Using Ambient Excitations (SESAME) guidelines, we can determine fundamental frequency and a minimum site amplification factor. We installed broadband seismometers throughout the Cal Poly Pomona campus, with an initial number of about 15 sites. The sites are approximately 50 to 150 meters apart and about two hours of waveforms were recorded at each site. We used the Geopsy software to make measurements of the peak frequency and the amplitude of the main peak from the spectral ratio. These two parameters have been determined to be estimates of fundamental frequency and a minimum site amplification factor, respectively. Based on the geological map from the U.S. Geological Survey (USGS) and our data collected from Cal Poly Pomona campus, our preliminary results suggest that the area of campus that is covered by alluvial fan material tends to have a single significant spectral peak with a fundamental frequency of ~1Hz and a minimum amplification factor of ~3.7. The minimum depth of the surface layer is about 56

  19. 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.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2016-08-01

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

  3. Structural Characterization of Human 8-Oxoguanine DNA Glycosylase Variants Bearing Active Site Mutations

    SciTech Connect

    Radom,C.; Banerjee, A.; Verdine, G.

    2007-01-01

    The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5 to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is 'hardwired'. Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F{sup *149}) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F{sup *292}) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

  4. Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation.

    PubMed

    Gao, Wenqing; Yang, Jieling; Liu, Wang; Wang, Yupeng; Shao, Feng

    2016-08-16

    Pyrin, encoded by the MEFV gene, is best known for its gain-of-function mutations causing familial Mediterranean fever (FMF), an autoinflammatory disease. Pyrin forms a caspase-1-activating inflammasome in response to inactivating modifications of Rho GTPases by various bacterial toxins or effectors. Pyrin-mediated innate immunity is unique in that it senses bacterial virulence rather than microbial molecules, but its mechanism of activation is unknown. Here we show that Pyrin was phosphorylated in bone marrow-derived macrophages and dendritic cells. We identified Ser-205 and Ser-241 in mouse Pyrin whose phosphorylation resulted in inhibitory binding by cellular 14-3-3 proteins. The two serines underwent dephosphorylation upon toxin stimulation or bacterial infection, triggering 14-3-3 dissociation, which correlated with Pyrin inflammasome activation. We developed antibodies specific for phosphorylated Ser-205 and Ser-241, which confirmed the stimuli-induced dephosphorylation of endogenous Pyrin. Mutational analyses indicated that both phosphorylation and signal-induced dephosphorylation of Ser-205/241 are important for Pyrin activation. Moreover, microtubule drugs, including colchicine, commonly used to treat FMF, effectively blocked activation of the Pyrin inflammasome. These drugs did not affect Pyrin dephosphorylation and 14-3-3 dissociation but inhibited Pyrin-mediated apoptosis-associated Speck-like protein containing CARD (ASC) aggregation. Our study reveals that site-specific (de)phosphorylation and microtubule dynamics critically control Pyrin inflammasome activation, illustrating a fine and complex mechanism in cytosolic immunity. PMID:27482109

  5. Acamprosate-responsive brain sites for suppression of ethanol intake and preference

    PubMed Central

    Brager, Allison; Prosser, Rebecca A.

    2011-01-01

    Acamprosate suppresses alcohol intake and craving in recovering alcoholics; however, the central sites of its action are unclear. To approach this question, brain regions responsive to acamprosate were mapped using acamprosate microimplants targeted to brain reward and circadian areas implicated in alcohol dependence. mPer2 mutant mice with nonfunctional mPer2, a circadian clock gene that gates endogenous timekeeping, were included, owing to their high levels of ethanol intake and preference. Male wild-type (WT) and mPer2 mutant mice received free-choice (15%) ethanol/water for 3 wk. The ethanol was withdrawn for 3 wk and then reintroduced to facilitate relapse. Four days before ethanol reintroduction, mice received bilateral blank or acamprosate-containing microimplants releasing ∼50 ng/day into reward [ventral tegmental (VTA), peduculopontine tegmentum (PPT), and nucleus accumbens (NA)] and circadian [intergeniculate leaflet (IGL) and suprachiasmatic nucleus (SCN)] areas. The hippocampus was also targeted. Circadian locomotor activity was measured throughout. Ethanol intake and preference were greater in mPer2 mutants than in wild-type (WT) mice (27 g·kg−1·day−1 vs. 13 g·kg−1·day−1 and 70% vs. 50%, respectively; both, P < 0.05). In WTs, acamprosate in all areas, except hippocampus, suppressed ethanol intake and preference (by 40–60%) during ethanol reintroduction. In mPer2 mutants, acamprosate in the VTA, PPT, and SCN suppressed ethanol intake and preference by 20–30%. These data are evidence that acamprosate's suppression of ethanol intake and preference are manifest through actions within major reward and circadian sites. PMID:21697518

  6. Solar activity, the QBO, and tropospheric responses

    NASA Technical Reports Server (NTRS)

    Tinsley, Brian A.; Brown, Geoffrey M.; Scherrer, Philip H.

    1989-01-01

    The suggestion that galactic cosmic rays (GCR) as modulated by the solar wind are the carriers of the component of solar variability that affects weather and climate has been discussed in the literature for 30 years, and there is now a considerable body of evidence that supports it. Variations of GCR occur with the 11 year solar cycle, matching the time scale of recent results for atmospheric variations, as modulated by the quasibiennial oscillation of equatorial stratospheric winds (the QBO). Variations in GCR occur on the time scale of centuries with a well defined peak in the coldest decade of the little ice age. New evidence is presented on the meteorological responses to GCR variations on the time scale of a few days. These responses include changes in the vertical temperature profile in the troposphere and lower stratosphere in the two days following solar flare related high speed plasma streams and associated GCR decreases, and in decreases in Vorticity Area Index (VAI) following Forbush decreases of GCR. The occurrence of correlations of GCR and meteorological responses on all three time scales strengthens the hypothesis of GCR as carriers of solar variability to the lower atmosphere. Both short and long term tropospheric responses are understandable as changes in the intensity of cyclonic storms initiated by mechanisms involving cloud microphysical and cloud electrification processes, due to changes in local ion production from changes in GCR fluxes and other high energy particles in the MeV to low GeV range. The nature of these mechanisms remains undetermined. Possible stratospheric wind (particularly QBO) effects on the transport of HNO3 and other constituents incorporated in cluster ions and possible condensation and freezing nuclei are considered as relevant to the long term variations.

  7. 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

  8. Macroscopic response in active nonlinear photonic crystals.

    PubMed

    Alagappan, Gandhi; John, Sajeev; Li, Er Ping

    2013-09-15

    We derive macroscopic equations of motion for the slowly varying electric field amplitude in three-dimensional active nonlinear optical nanostructures. We show that the microscopic Maxwell equations and polarization dynamics can be simplified to a macroscopic one-dimensional problem in the direction of group velocity. For a three-level active material, we derive the steady-state equations for normal mode frequency, threshold pumping, nonlinear Bloch mode amplitude, and lasing in photonic crystals. Our analytical results accurately recapture the results of exact numerical methods. PMID:24104802

  9. Rotor Flapping Response to Active Control

    NASA Technical Reports Server (NTRS)

    Nguyen, Khanh; Johnson, Wayne

    2004-01-01

    Rotor active control using higher harmonic blade pitch has been proposed as a means to reduce both rotor radiated noise and airframe vibration and to enhance rotor performance. The higher harmonic input, however, can affect rotor thrust and cyclic flapping - the basic trim characteristics of the rotor. Some of the trim changes can negate the active control benefits. For example, wind tunnel test results of a full scale BO-105 rotor with individual-blade control indicate some rotor performance improvements, accompanied with changes in rotor trim, using two-per-rev blade pitch input. The observed performance benefits could therefore be a simple manifestation of the trim change rather than an efficient redistribution of the rotor airloads. More recently, the flight test of the BO-105 helicopter equip,ped with individual-blade-control actuators also reported trim changes whenever the two-per-rev blade pitch for noise reduction was activated. The pilot had to adjust the trim control to maintain the aircraft under a constant flight path. These two cases highlight the, importance of trim considerations in the application of active control to rotorcraft.

  10. The unpredictability of prolonged activation of stress response pathways

    PubMed Central

    Lamech, Lilian T.

    2015-01-01

    In response to stress, cellular compartments activate signaling pathways that mediate transcriptional programs to promote survival and reestablish homeostasis. Manipulation of the magnitude and duration of the activation of stress responses has been proposed as a strategy to prevent or repair the damage associated with aging or degenerative diseases. However, as these pathways likely evolved to respond specifically to transient perturbations, the unpredictability of prolonged activation should be considered. PMID:26101215

  11. Geophysical Signatures of Microbial Activity at Hydrocarbon Contaminated Sites: A Review

    NASA Astrophysics Data System (ADS)

    Atekwana, Estella A.; Atekwana, Eliot A.

    2010-03-01

    Microorganisms participate in a variety of geologic processes that alter the chemical and physical properties of their environment. Understanding the geophysical signatures of microbial activity in the environment has resulted in the development of a new sub-discipline in geophysics called “biogeophysics”. This review focuses primarily on literature pertaining to biogeophysical signatures of sites contaminated by light non-aqueous phase liquids (LNAPL), as these sites provide ideal laboratories for investigating microbial-geophysical relationships. We discuss the spatial distribution and partitioning of LNAPL into different phases because the physical, chemical, and biological alteration of LNAPL and the subsequent impact to the contaminated environment is in large part due to its distribution. We examine the geophysical responses at contaminated sites over short time frames of weeks to several years when the alteration of the LNAPL by microbial activity has not occurred to a significant extent, and over the long-term of several years to decades, when significant microbial degradation of the LNAPL has occurred. A review of the literature suggests that microbial processes profoundly alter the contaminated environment causing marked changes in the petrophysical properties, mineralogy, solute concentration of pore fluids, and temperature. A variety of geophysical techniques such as electrical resistivity, induced polarization, electromagnetic induction, ground penetrating radar, and self potential are capable of defining the contaminated zones because of the new physical properties imparted by microbial processes. The changes in the physical properties of the contaminated environment vary spatially because microbial processes are controlled by the spatial distribution of the contaminant. Geophysical studies must consider the spatial variations in the physical properties during survey design, data analysis, and interpretation. Geophysical data interpretation from

  12. 40 CFR 300.440 - Procedures for planning and implementing off-site response actions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 28 2011-07-01 2011-07-01 false Procedures for planning and implementing off-site response actions. 300.440 Section 300.440 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SUPERFUND, EMERGENCY PLANNING, AND COMMUNITY RIGHT-TO-KNOW PROGRAMS NATIONAL OIL AND HAZARDOUS SUBSTANCES...

  13. Preliminary evaluation of the Accident Response Mobile Manipulation System for accident site salvage operations

    SciTech Connect

    Trujillo, J.M.; Morse, W.D.; Jones, D.P.

    1994-10-01

    This paper describes and evaluates operational experiences with the Accident Response Mobile Manipulation System (ARMMS) during simulated accident site salvage operations which might involve nuclear weapons. The ARMMS is based upon a teleoperated mobility platform with two Schilling Titan 7F Manipulators.

  14. Study of Site Response in the Seattle and Tacoma Basins, Washington, Using Spectral Ratio Methods

    NASA Astrophysics Data System (ADS)

    Keshvardoost, R.; Wolf, L. W.

    2014-12-01

    Sedimentary basins are known to have a pronounced influence on earthquake-generated ground motions, affecting both predominant frequencies and wave amplification. These site characteristics are important elements in estimating ground shaking and seismic hazard. In this study, we use three-component broadband and strong motion seismic data from three recent earthquakes to determine site response characteristics in the Seattle and Tacoma basins, Washington. Resonant frequencies and relative amplification of ground motions were determined using Fourier spectral ratios of velocity and acceleration records from the 2012 Mw 6.1 Vancouver Island earthquake, the 2012 Mw 7.8 Queen Charlotte Island earthquake, and the 2014 Mw 6.6 Vancouver Island earthquake. Recordings from sites within and adjacent to the Seattle and Tacoma basins were selected for the study based on their signal to noise ratios. Both the Standard Spectral Ratio (SSR) and the Horizontal-to-Vertical Spectral Ratio (HVSR) methods were used in the analysis, and results from each were compared to examine their agreement and their relation to local geology. Although 57% of the sites (27 out of 48) exhibited consistent results between the two methods, other sites varied considerably. In addition, we use data from the Seattle Liquefaction Array (SLA) to evaluate the site response at 4 different depths. Results indicate that resonant frequencies remain the same at different depths but amplification decreases significantly over the top 50 m.

  15. Age and skeletal sites affect BMP-2 responsiveness of human bone marrow stromal cells.

    PubMed

    Osyczka, Anna Maria; Damek-Poprawa, Monika; Wojtowicz, Aleksandra; Akintoye, Sunday O

    2009-01-01

    Bone marrow stromal cells (BMSCs) contain osteoprogenitors responsive to stimulation by osteogenic growth factors like bone morphogenetic proteins (BMPs). When used as grafts, BMSCs can be harvested from different skeletal sites such as axial, appendicular, and orofacial bones, but the lower therapeutic efficacy of BMPs on BMSCs-responsiveness in humans compared to animal models may be due partly to effects of skeletal site and age of donor. We previously reported superior differentiation capacity and osteogenic properties of orofacial BMSCs relative to iliac crest BMSCs in same individuals. This study tested the hypothesis that recombinant human BMP-2 (rhBMP-2) stimulates human BMSCs differently based on age and skeletal site of harvest. Adult maxilla, mandible, and iliac crest BMSCs from same individuals and pediatric iliac crest BMSCs were comparatively assessed for BMP-2 responsiveness under serum-containing and serum-free insulin-supplemented culture conditions. Adult orofacial BMSCs were more BMP-2-responsive than iliac crest BMSCs based on higher gene transcripts of alkaline phosphatase, osteopontin, and osteogenic transcription factors MSX-2 and Osterix in serum-free insulin-containing medium. Pediatric iliac crest BMSCs were more responsive to rhBMP-2 than adult iliac crest BMSCs based on higher expression of alkaline phosphatase and osteopontin in serum-containing medium. Unlike orofacial BMSCs, MSX-2 and Osterix transcripts were similarly expressed by adult and pediatric iliac crest BMSCs in response to rhBMP-2. These data demonstrate that age and skeletal site-specific differences exist in BMSC osteogenic responsiveness to BMP-2 stimulation and suggest that MSX-2 and Osterix may be potential regulatory transcription factors in BMP-mediated osteogenesis of adult orofacial cells. PMID:19637063

  16. Age and Skeletal Sites Affect BMP-2 Responsiveness of Human Bone Marrow Stromal Cells

    PubMed Central

    Osyczka, Anna M.; Damek-Poprawa, Monika; Wojtowicz, Aleksandra; Akintoye, Sunday O.

    2010-01-01

    Bone marrow stromal cells (BMSCs) contain osteoprogenitors responsive to stimulation by osteogenic growth factors like bone morphogenetic proteins (BMPs). When used as grafts, BMSCs can be harvested from different skeletal sites such as axial, appendicular and orofacial bones, but the lower therapeutic efficacy of BMPs on BMSCs-responsiveness in humans compared to animal models may be partly due to effects of skeletal site and age of donor. We previously reported superior differentiation capacity and osteogenic properties of orofacial BMSCs relative to iliac crest BMSCs in same individuals. This study tested the hypothesis that recombinant human BMP-2 (rhBMP-2) stimulates human BMSCs differently based on age and skeletal site of harvest. Adult maxilla, mandible and iliac crest BMSCs from same individuals and pediatric iliac crest BMSCs were comparatively assessed for BMP-2 responsiveness under serum-containing and serum-free insulin-supplemented culture conditions. Adult orofacial BMSCs were more BMP-2-responsive than iliac crest BMSCs based on higher gene transcripts of alkaline phosphatase, osteopontin and osteogenic transcription factors MSX-2 and Osterix in serum-free insulin-containing medium. Pediatric iliac crest BMSCs were more responsive to rhBMP-2 than adult iliac crest BMSCs based on higher expression of alkaline phosphatase and osteopontin in serum-containing medium. Unlike orofacial BMSCs, MSX-2 and Osterix transcripts were similarly expressed by adult and pediatric iliac crest BMSCs in response to rhBMP-2. These data demonstrate that age and skeletal site-specific differences exist in BMSC osteogenic responsiveness to BMP-2 stimulation and suggest that MSX-2 and Osterix may be potential regulatory transcription factors in BMP-mediated osteogenesis of adult orofacial cells. PMID:19637063

  17. Dynamics of telomerase activity in response to acute psychological stress

    PubMed Central

    Epel, Elissa S.; Lin, Jue; Dhabhar, Firdaus S.; Wolkowitz, Owen M.; Puterman, E; Karan, Lori; Blackburn, Elizabeth H.

    2010-01-01

    Telomerase activity plays an essential role in cel0l survival, by lengthening telomeres and promoting cell growth and longevity. It is now possible to quantify the low levels of telomerase activity in human leukocytes. Low basal telomerase activity has been related to chronic stress in people and to chronic glucocorticoid exposure in vitro. Here we test whether leukocyte telomerase activity changes under acute psychological stress. We exposed 44 elderly women, including 22 high stress dementia caregivers and 22 matched low stress controls, to a brief laboratory psychological stressor, while examining changes in telomerase activity of peripheral blood mononuclear cells (PBMC). At baseline, caregivers had lower telomerase activity levels than controls, but during stress telomerase activity increased similarly in both groups. Across the entire sample, subsequent telomerase activity increased by 18% one hour after the end of the stressor (p<0.01). The increase in telomerase activity was independent of changes in numbers or percentages of monocytes, lymphocytes, and specific T cell types, although we cannot fully rule out some potential contribution from immune cell redistribution in the change in telomerase activity. Telomerase activity increases were associated with greater cortisol increases in response to the stressor. Lastly, psychological response to the tasks (greater threat perception) was also related to greater telomerase activity increases in controls. These findings uncover novel relationships of dynamic telomerase activity with exposure to an acute stressor, and with two classic aspects of the stress response -- perceived psychological stress and neuroendocrine (cortisol) responses to the stressor. PMID:20018236

  18. Near-Infrared Light-Responsive Hydrogel for Specific Recognition and Photothermal Site-Release of Circulating Tumor Cells.

    PubMed

    Lv, Song-Wei; Liu, Ya; Xie, Min; Wang, Jing; Yan, Xue-Wei; Li, Zhen; Dong, Wei-Guo; Huang, Wei-Hua

    2016-06-28

    Isolation of single circulating tumor cells (CTCs) from patients is a very challenging technique that may promote the process of individualized antitumor therapies. However, there exist few systems capable of highly efficient capture and release of single CTCs with high viability for downstream analysis and culture. Herein, we designed a near-infrared (NIR) light-responsive substrate for highly efficient immunocapture and biocompatible site-release of CTCs by a combination of the photothermal effect of gold nanorods (GNRs) and a thermoresponsive hydrogel. The substrate was fabricated by imprinting target cancer cells on a GNR-pre-embedded gelatin hydrogel. Micro/nanostructures generated by cell imprinting produce artificial receptors for cancer cells to improve capture efficiency. Temperature-responsive gelatin dissolves rapidly at 37 °C; this allows bulk recovery of captured CTCs at physiological temperature or site-specific release of single CTCs by NIR-mediated photothermal activation of embedded GNRs. Furthermore, the system has been applied to capture, individually release, and genetically analyze CTCs from the whole blood of cancer patients. The multifunctional NIR-responsive platform demonstrates excellent performance in capture and site-release of CTCs with high viability, which provides a robust and versatile means toward individualized antitumor therapies and also shows promising potential for dynamically manipulating cell-substrate interactions in vitro. PMID:27299807

  19. Measurement of solvation responses at multiple sites in a globular protein.

    PubMed

    Abbyad, Paul; Shi, Xinghua; Childs, William; McAnaney, Tim B; Cohen, Bruce E; Boxer, Steven G

    2007-07-19

    Proteins respond to electrostatic perturbations through complex reorganizations of their charged and polar groups, as well as those of the surrounding media. These solvation responses occur both in the protein interior and on its surface, though the exact mechanisms of solvation are not well understood, in part because of limited data on the solvation responses for any given protein. Here, we characterize the solvation kinetics at sites throughout the sequence of a small globular protein, the B1 domain of streptococcal protein G (GB1), using the synthetic fluorescent amino acid Aladan. Aladan was incorporated into seven different GB1 sites, and the time-dependent Stokes shift was measured over the femtosecond to nanosecond time scales by fluorescence upconversion and time-correlated single photon counting. The seven sites range from buried within the protein core to fully solvent-exposed on the protein surface, and are located on different protein secondary structures including beta-sheets, helices, and loops. The dynamics in the protein sites were compared against the free fluorophore in buffer. All protein sites exhibited an initial, ultrafast Stokes shift on the subpicosecond time scale similar to that observed for the free fluorophore, but smaller in magnitude. As the probe is moved from the surface to more buried sites, the dynamics of the solvation response become slower, while no clear correlation between dynamics and secondary structure is observed. We suggest that restricted movements of the surrounding protein residues give rise to the observed long time dynamics and that such movements comprise a large portion of the protein's solvation response. The proper treatment of dynamic Stokes shift data when the time scale for solvation is comparable to the fluorescence lifetime is discussed. PMID:17592867

  20. Measurement of Solvation Responses at Multiple Sites in a Globular Protein

    PubMed Central

    Abbyad, Paul; Shi, Xinghua; Childs, William; McAnaney, Tim B.; Cohen, Bruce E.; Boxer, Steven G.

    2008-01-01

    Proteins respond to electrostatic perturbations through complex reorganizations of their charged and polar groups, as well as those of the surrounding media. These solvation responses occur both in the protein interior and on its surface, though the exact mechanisms of solvation are not well understood, in part because of limited data on the solvation responses for any given protein. Here, we characterize the solvation kinetics at sites throughout the sequence of a small globular protein, the B1 domain of streptococcal protein G (GB1), using the synthetic fluorescent amino acid Aladan. Aladan was incorporated into seven different GB1 sites, and the time-dependent Stokes shift measured over the femtosecond to nanosecond timescales by fluorescence upconversion and time-correlated single photon counting. The seven sites range from buried within the protein core to fully solvent-exposed on the protein surface, and are located on different protein secondary structures including β-sheets, helices and loops. The dynamics in the protein sites were compared against the free fluorophore in buffer. All protein sites exhibited an initial, ultra-fast Stokes shift on the sub-picosecond timescale similar to that observed for the free fluorophore, but smaller in magnitude. As the probe is moved from the surface to more buried sites, the dynamics of the solvation response become slower, while no clear correlation between dynamics and secondary structure is observed. We suggest that restricted movements of the surrounding protein residues give rise to the observed long time dynamics and that such movements comprise a large portion of the protein’s solvation response. The proper treatment of dynamic Stokes shift data when the timescale for solvation is comparable to the fluorescence lifetime is discussed. PMID:17592867

  1. Seismic Wave Amplification in Las Vegas: Site Response and Empirical Estimates of Ground Motion

    NASA Astrophysics Data System (ADS)

    Rodgers, A.; McCallen, D.; Tkalcic, H.; Wagoner, J.; Louie, J.; Anderson, J.; Luke, B.; Snelson, C.; Taylor, W.

    2004-12-01

    This presentation will summarize a multidisciplinary effort to understand seismic wave amplification in Las Vegas Valley. The project involves weak motion recording and analysis, geotechnical and seismic refraction field studies, geologic and lithologic interpretation and model building. We will provide a brief overview of the project, then focus on specifics of seismic wave amplification including observations and interpretations. We analyzed recordings of nuclear explosions from the Nevada Test Site (NTS) and regional earthquakes to estimate site response in Las Vegas. An empirical transfer function method was used to transform ground motion time-series at one (reference) station to other stations, using frequency dependent site response curves in the band 0.2-5.0 Hz. The method transforms the time-series to the frequency domain by Fast Fourier transform, multiplies the amplitude spectrum by the site response curve and inverse FFT's back to the time domain. The approach is validated by the ability to predict horizontal component S-wave ground motion measures, such as peak and rms ground velocities and accelerations. We then can provide empirical estimates of ground motion for a wider distribution of sites in Las Vegas. Frequency dependent amplifications (site response) and peak ground motions are strongly correlated with measures of shallow shear-wave (geotechnical) velocities. Details of the geotechnical measurements and models will be presented in a companion presentation. This work was performed under the auspices of the U.S. Department of Energy by University of California Lawrence Livermore National Laboratory under contract No. W-7405-Eng-48.

  2. Natural resource management activities at the Savannah River Site. Environmental Assessment

    SciTech Connect

    Not Available

    1993-07-01

    This environmental assessment (EA) reviews the environmental consequences of ongoing natural resource management activities on the Savannah River Site (SRS). Appendix A contains the Natural Resources Management Plant (NRMP). While several SRS organizations have primary responsibilities for different elements of the plan, the United States Department of Agriculture (USDA), Forest Service, Savannah River Forest Station (SRFS) is responsible for most elements. Of the river scenarios defined in 1985, the High-Intensity Management alternative established the upper bound of environmental consequences; it represents a more intense level of resource management than that being performed under current resource management activities. This alternative established compliance mechanisms for several natural resource-related requirements and maximum practical timber harvesting. Similarly, the Low-Intensity Management alternative established the lower bound of environmental consequences and represents a less intense level of resource management than that being performed under current resource management activities. This alternative also established compliance mechanisms, but defined a passively managed natural area. The Proposed Action of this EA describes the current level of multiple-natural resource management. This EA reviews the proposed action, and the high and low intensity alternative scenarios.

  3. Demographic responses of amphibians to wetland restoration in Carolina bays on the Savannah River Site.

    SciTech Connect

    Kinkead, Karen E.

    2004-09-30

    This project studied the effects of wetland restoration on amphibian populations. These wetlands were Carolina bays located on the Savannah River Site, located near Aiken, S.C. The Savannah River Site is a National Environmental Research Park owned and operated by the U.S. Department of Energy. The study sites included three reference bays (functionally intact), three control bays (with active drainage ditches), six treatment bays (restored during 2001), and four bays near two of the treatment bays (in effect creating two metapopulations).

  4. Inhibitory activity of the equine infectious anemia virus major 5' splice site in the absence of Rev.

    PubMed Central

    Tan, W; Schalling, M; Zhao, C; Luukkonen, M; Nilsson, M; Fenyö, E M; Pavlakis, G N; Schwartz, S

    1996-01-01

    The major 5' splice site of equine infectious anemia virus (EIAV) conforms to the consensus 5' splice site in eight consecutive positions and is located immediately upstream of the gag AUG. Our results show that the presence of this 5' splice site on the EIAV gag mRNA decreases Gag production 30- to 60-fold. This is caused by inefficient nuclear mRNA export and inefficient mRNA utilization. Inhibition could be overcome by providing human immunodeficiency virus type 1 Rev/Rev-responsive element, human T-cell leukemia virus type 1 Rex/Rex-responsive element, or simian retrovirus type 1 constitutive transport element. In addition, inhibition could be abolished by introducing single point mutations in the 5' splice site or by moving the 5' splice site away from its natural position immediately upstream of the gag AUG. This demonstrates that both maintenance of a perfect consensus 5' splice site and its proper location on the mRNA are important for inhibitory activity of the EIAV major 5' splice site. PMID:8648699

  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. Pulmonary infection with influenza A virus induces site-specific germinal center and T follicular helper cell responses.

    PubMed

    Boyden, Alexander W; Legge, Kevin L; Waldschmidt, Thomas J

    2012-01-01

    Protection from influenza A virus (IAV) challenge requires switched, high affinity Abs derived from long-lived memory B cells and plasma cells. These B cell subsets are generated in germinal centers (GCs), hallmark structures of T helper cell-driven B cell immunity. A full understanding of the GC reaction after respiratory IAV infection is lacking, as is the characterization of T follicular helper (T(FH)) cells that support GCs. Here, GC B cell and T(FH) cell responses were studied in mice following pulmonary challenge with IAV. Marked GC reactions were induced in draining lymph nodes (dLNs), lung, spleen and nasal-associated lymphoid tissue (NALT), although the magnitude and kinetics of the response was site-specific. Examination of switching within GCs demonstrated IgG2(+) cells to compose the largest fraction in dLNs, lung and spleen. IgA(+) GC B cells were infrequent in these sites, but composed a significant subset of the switched GC population in NALT. Further experiments demonstrated splenectomized mice to withstand a lethal recall challenge, suggesting the spleen to be unnecessary for long-term protection in spite of strong GC responses in this organ. Final studies showed that T(FH) cell numbers were highest in dLNs and spleen, and peaked in all sites prior to the height of the GC reaction. T(FH) cells purified from dLNs generated IL-21 and IFNγ upon activation, although CD4(+)CXCR5(-) T effector cells produced higher levels of all cytokines. Collectively, these findings reveal respiratory IAV infection to induce strong T helper cell-driven B cell responses in various organs, with each site displaying unique attributes. PMID:22792401

  7. 78 FR 33908 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-05

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

  8. 77 FR 39508 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-03

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

  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. Mutation of conserved active-site threonine residues in creatine kinase affects autophosphorylation and enzyme kinetics.

    PubMed Central

    Stolz, Martin; Hornemann, Thorsten; Schlattner, Uwe; Wallimann, Theo

    2002-01-01

    Muscle-type creatine kinase (MM-CK) is a member of an isoenzyme family with key functions in cellular energetics. It has become a matter of debate whether the enzyme is autophosphorylated, as reported earlier [Hemmer, Furter-Graves, Frank, Wallimann and Furter (1995) Biochim. Biophys. Acta 1251, 81-90], or exclusively nucleotidylated. In the present paper, we demonstrate unambiguously that CK is indeed autophosphorylated. However, this autophosphorylation is not solely responsible for the observed microheterogeneity of MM-CK on two-dimensional isoelectric focusing gels. Using phosphoamino-acid analysis of (32)P-labelled CK isoforms, phosphothreonine (P-Thr) residues were identified as the only product of autophosphorylation for all CK isoenzymes. The phosphorylated residues in chicken MM-CK were allocated to a region in the vicinity of the active site, where five putative phosphorylation sites were identified. Site-directed threonine-valine-replacement mutants reveal that autophosphorylation is not specific for one particular residue but occurs at all examined threonine residues. The enzyme kinetic parameters indicate that the autophosphorylation of CK exerts a modulatory effect on substrate binding and the equilibrium constant, rather than on the catalytic mechanism itself. PMID:11964180

  11. 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)

  12. 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.

  13. Ground motion in Anchorage, Alaska, from the 2002 Denali fault earthquake: Site response and Displacement Pulses

    USGS Publications Warehouse

    Boore, D.M.

    2004-01-01

    Data from the 2002 Denali fault earthquake recorded at 26 sites in and near Anchorage, Alaska, show a number of systematic features important in studies of site response and in constructing long-period spectra for use in earthquake engineering. The data demonstrate that National Earthquake Hazards Reduction Program (NEHRP) site classes are a useful way of grouping stations according to site amplification. In general, the sites underlain by lower shear-wave velocities have higher amplification. The amplification on NEHRP class D sites exceeds a factor of 2 relative to an average of motions on class C sites. The amplifications are period dependent. They are in rough agreement with those from previous studies, but the new data show that the amplifications extend to at least 10 sec, periods longer than considered in previous studies. At periods longer than about 14 sec, all sites have motion of similar amplitude, and the ground displacements are similar in shape, polarization, and amplitude for all stations. The displacement ground motion is dominated by a series of four pulses, which are associated with the three subevents identified in inversion studies (the first pulse is composed of P waves from the first subevent). Most of the high-frequency ground motion is associated with the S waves from subevent 1. The pulses from subevents 1 and 2, with moment releases corresponding to M 7.1 and 7.0, are similar to the pulse of displacement radiated by the M 7.1 Hector Mine earthquake. The signature from the largest subevent (M 7.6) is more subdued than those from the first two subevents. The two largest pulses produce response spectra with peaks at a period of about 15 sec. The spectral shape at long periods is in good agreement with the recent 2003 NEHRP code spectra but is in poor agreement with the shape obtained from Eurocode 8.

  14. Host-Guest Binding-Site-Tunable Self-Assembly of Stimuli-Responsive Supramolecular Polymers.

    PubMed

    Yao, Hao; Qi, Miao; Liu, Yuyang; Tian, Wei

    2016-06-13

    Despite the remarkable progress made in controllable self-assembly of stimuli-responsive supramolecular polymers (SSPs), a basic issue that has not been consideration to date is the essential binding site. The noncovalent binding sites, which connect the building blocks and endow supramolecular polymers with their ability to respond to stimuli, are expected to strongly affect the self-assembly of SSPs. Herein, the design and synthesis of a dual-stimuli thermo- and photoresponsive Y-shaped supramolecular polymer (SSP2) with two adjacent β-cyclodextrin/azobenzene (β-CD/Azo) binding sites, and another SSP (SSP1) with similar building blocks, but only one β-CD/Azo binding site as a control, are described. Upon gradually increasing the polymer solution temperature or irradiating with UV light, SSP2 self-assemblies with a higher binding-site distribution density; exhibits a flower-like morphology, smaller size, and more stable dynamic aggregation process; and greater controllability for drug-release behavior than those observed with SSP1 self-assemblies. The host-guest binding-site-tunable self-assembly was attributed to the positive cooperativity generated among adjacent binding sites on the surfaces of SSP2 self-assemblies. This work is beneficial for precisely controlling the structural parameters and controlled release function of SSP self-assemblies. PMID:27167577

  15. 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…

  16. Near Surface Characterization and Estimated Site-Response at POLARIS Seismograph Stations in Southern Ontario

    NASA Astrophysics Data System (ADS)

    Murphy, C.; Eaton, D.

    2002-12-01

    Shallow seismic and ground penetrating radar (GPR) surveys have been collected in southern Ontario, Canada near current and planned seismograph stations for the POLARIS network. All five of the sites are characterized by glaciolacustrine overburden, consisting of silt, sand, and/or clay, on the order of a few metres to ten's of metres. Bedrock at all of the sites is mapped as Paleozoic limestone and shale, and is located at depths ranging from a few metres to ten's of metres. The objectives of these surveys are to obtain shallow velocity models (P and S-wave) at each site for earthquake site-response determination, and to investigate the utility of GPR data for siting underground vaults. The seismic surveys were acquired using 24- or 48-channel systems, with several different sources (an in-hole shotgun or a sledge hammer). The use of a twelve pound sledge hammer to strike a steel I-beam perpendicular to its length, oriented in-line with the geophone spread, was found to produce strong SH waves. In order to constrain the velocities at both very shallow (< 4 m) and deeper (4-30 m) depth intervals, two geophone spreads were collected, one with a one metre geophone spacing and another with a five metre geophone spacing. Velocity models derived by refraction analysis provide the basis for classification of each site according to the NEHRP scheme. In addition, a comparison of the response at each site is being undertaken by visco-elastic finite-difference modeling of the ground motion induced by incident wavefields of various types (both regional and teleseismic). Where possible, these results are compared with empirical site-response determined from regional events using the H/V method. The site response obtained for each station will be used in the future for calibration of the POLARIS network. In one case, the GPR data led to the discovery of a probable underground drainage channel that would have been an inappropriate location for a seismograph vault.

  17. Site Response Analysis of the Monte Po Hill in the City of Catania

    SciTech Connect

    Cavallaro, A.; Ferraro, A.; Grasso, S.; Maugeri, M.

    2008-07-08

    The Monte Po Hill is located in the North-eastern part of the city of Catania; this area is prone to high seismic risk due to the presence of several constructions, including a school, in the vicinity of a toe of a slope, characterized by precarious stability conditions. For site characterization of the soil, deep site investigations have been undertaken. Borings and dynamic in situ tests have been performed. Among them Down-Hole (D-H) and Seismic Dilatometer Marchetti Tests (SDMT) have been carried out, with the aim to evaluate the soil profile of shear waves velocity (Vs). The Seismic Dilatometer Marchetti Tests were performed up to a depth of 15 meters. The results show a very detailed and stable shear waves profile. The shear waves profiles obtained by SDMT compare well with other in situ tests. Synthetic seismograms have been drawn for the site long a set of receivers placed at different depths, starting from the surface up to almost 40 m. After evaluating the synthetic accelerograms at the bedrock, the ground response analysis at the surface, in terms of time history and response spectra, has been obtained by a 1-D non-linear model. In particular the study has regarded the evaluation of site effects in correspondence of the site, to which corresponds a different value of the Seismic Geotechnical Hazard. In the beginning of 2007 a seismic station has been also located into the school building, with the aim of recording seismic events. Seismograms obtained by the seismic station have been also used to evaluate the ground response analysis at the surface. In the beginning of 2007 a seismic station has been also located into the school building, with the aim of recording seismic events. Seismograms obtained by the seismic station have been also used to evaluate the ground response analysis at the surface. Finally the 1-D computer code EERA was also used to model the equivalent-linear earthquake site response analyses of layered soil deposits of the hill. The

  18. Ionospheric Response Due to Seismic Activity

    NASA Astrophysics Data System (ADS)

    Sharma, Dinesh Kumar

    2016-07-01

    Signatures of the seismic activity in the ionospheric F2 region have been studied by analyzing the measurement of electron and ion temperatures during the occurrence of earthquake. The ionospheric electron and ion temperatures data recorded by the RPA payload aboard the Indian SROSS-C2 satellite during the period from January 1995 to December 2000 were used for the altitude range 430-630 km over Indian region. The normal day's electron and ion temperatures have been compared to the temperatures recorded during the seismic activity. The details of seismic events were obtained from USGS earthquake data information website. It has been found that the average electron temperature is enhanced during the occurrence of earthquakes by 1.2 to 1.5 times and this enhancement was for ion temperature ranging from 1.1to 1.3 times over the normal day's average temperatures. The above careful quantitative analysis of ionospheric electron and ion temperatures data shows the consistent enhancement in the ionospheric electron and ion temperatures. It is expected that the seismogenic vertical electrical field propagates up to the ionospheric heights and induces Joule heating that may cause the enhancement in ionospheric temperatures.

  19. 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.

  20. Unconventional Human T Cells Accumulate at the Site of Infection in Response to Microbial Ligands and Induce Local Tissue Remodeling

    PubMed Central

    Liuzzi, Anna Rita; Kift-Morgan, Ann; Lopez-Anton, Melisa; Friberg, Ida M.; Zhang, Jingjing; Brook, Amy C.; Roberts, Gareth W.; Donovan, Kieron L.; Colmont, Chantal S.; Toleman, Mark A.; Bowen, Timothy; Johnson, David W.; Topley, Nicholas; Moser, Bernhard; Fraser, Donald J.

    2016-01-01

    The antimicrobial responsiveness and function of unconventional human T cells are poorly understood, with only limited access to relevant specimens from sites of infection. Peritonitis is a common and serious complication in individuals with end-stage kidney disease receiving peritoneal dialysis. By analyzing local and systemic immune responses in peritoneal dialysis patients presenting with acute bacterial peritonitis and monitoring individuals before and during defined infectious episodes, our data show that Vγ9/Vδ2+ γδ T cells and mucosal-associated invariant T cells accumulate at the site of infection with organisms producing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and vitamin B2, respectively. Such unconventional human T cells are major producers of IFN-γ and TNF-α in response to these ligands that are shared by many microbial pathogens and affect the cells lining the peritoneal cavity by triggering local inflammation and inducing tissue remodeling with consequences for peritoneal membrane integrity. Our data uncover a crucial role for Vγ9/Vδ2 T cells and mucosal-associated invariant T cells in bacterial infection and suggest that they represent a useful predictive marker for important clinical outcomes, which may inform future stratification and patient management. These findings are likely to be applicable to other acute infections where local activation of unconventional T cells contributes to the antimicrobial inflammatory response. PMID:27527598

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2015-12-29

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

  3. Snoring Sounds Predict Obstruction Sites and Surgical Response in Patients with Obstructive Sleep Apnea Hypopnea Syndrome.

    PubMed

    Lee, Li-Ang; Lo, Yu-Lun; Yu, Jen-Fang; Lee, Gui-She; Ni, Yung-Lun; Chen, Ning-Hung; Fang, Tuan-Jen; Huang, Chung-Guei; Cheng, Wen-Nuan; Li, Hsueh-Yu

    2016-01-01

    Snoring sounds generated by different vibrators of the upper airway may be useful indicators of obstruction sites in patients with obstructive sleep apnea hypopnea syndrome (OSAHS). This study aimed to investigate associations between snoring sounds, obstruction sites, and surgical responses (≥50% reduction in the apnea-hypopnea index [AHI] and <10 events/hour) in patients with OSAHS. This prospective cohort study recruited 36 OSAHS patients for 6-hour snoring sound recordings during in-lab full-night polysomnography, drug-induced sleep endoscopy (DISE), and relocation pharyngoplasty. All patients received follow-up polysomnography after 6 months. Fifteen (42%) patients with at least two complete obstruction sites defined by DISE were significantly, positively associated with maximal snoring sound intensity (40-300 Hz; odds ratio [OR], 1.25, 95% confidence interval [CI] 1.05-1.49) and body mass index (OR, 1.48, 95% CI 1.02-2.15) after logistic regression analysis. Tonsil obstruction was significantly, inversely correlated with mean snoring sound intensity (301-850 Hz; OR, 0.84, 95% CI 0.74-0.96). Moreover, baseline tonsil obstruction detected by either DISE or mean snoring sound intensity (301-850 Hz), and AHI could significantly predict the surgical response. Our findings suggest that snoring sound detection may be helpful in determining obstruction sites and predict surgical responses. PMID:27471038

  4. Snoring Sounds Predict Obstruction Sites and Surgical Response in Patients with Obstructive Sleep Apnea Hypopnea Syndrome

    PubMed Central

    Lee, Li-Ang; Lo, Yu-Lun; Yu, Jen-Fang; Lee, Gui-She; Ni, Yung-Lun; Chen, Ning-Hung; Fang, Tuan-Jen; Huang, Chung-Guei; Cheng, Wen-Nuan; Li, Hsueh-Yu

    2016-01-01

    Snoring sounds generated by different vibrators of the upper airway may be useful indicators of obstruction sites in patients with obstructive sleep apnea hypopnea syndrome (OSAHS). This study aimed to investigate associations between snoring sounds, obstruction sites, and surgical responses (≥50% reduction in the apnea-hypopnea index [AHI] and <10 events/hour) in patients with OSAHS. This prospective cohort study recruited 36 OSAHS patients for 6-hour snoring sound recordings during in-lab full-night polysomnography, drug-induced sleep endoscopy (DISE), and relocation pharyngoplasty. All patients received follow-up polysomnography after 6 months. Fifteen (42%) patients with at least two complete obstruction sites defined by DISE were significantly, positively associated with maximal snoring sound intensity (40–300 Hz; odds ratio [OR], 1.25, 95% confidence interval [CI] 1.05–1.49) and body mass index (OR, 1.48, 95% CI 1.02–2.15) after logistic regression analysis. Tonsil obstruction was significantly, inversely correlated with mean snoring sound intensity (301–850 Hz; OR, 0.84, 95% CI 0.74–0.96). Moreover, baseline tonsil obstruction detected by either DISE or mean snoring sound intensity (301–850 Hz), and AHI could significantly predict the surgical response. Our findings suggest that snoring sound detection may be helpful in determining obstruction sites and predict surgical responses. PMID:27471038

  5. The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.

    PubMed Central

    Bahou, W F; Coller, B S; Potter, C L; Norton, K J; Kutok, J L; Goligorsky, M S

    1993-01-01

    A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha

  6. P-glycoprotein activity and biological response

    SciTech Connect

    Vaalburg, W. . E-mail: w.vaalburg@pet.umcg.nl; Hendrikse, N.H.; Elsinga, P.H.; Bart, J.; Waarde, A. van

    2005-09-01

    P-glycoprotein (P-gp) is a transmembrane drug efflux pump encoded by the MDR-1 gene in humans. Most likely P-gp protects organs against endogenous and exogenous toxins by extruding toxic compounds such as chemotherapeutics and other drugs. Many drugs are substrates for P-gp. Since P-gp is also expressed in the blood-brain barrier, P-gp substrates reach lower concentrations in the brain than in P-gp-negative tissues. Failure of response to chemotherapy of malignancies can be due to intrinsic or acquired drug resistance. Many tumors are multidrug resistant (MDR); resistant to several structurally unrelated chemotherapeutic agents. Several mechanisms are involved in MDR of which P-gp is studied most extensively. P-gp extrudes drugs out of tumor cells resulting in decreased intracellular drug concentrations, leading to the MDR phenotype. Furthermore, the MDR-1 gene exhibits several single nucleotide polymorphisms, some of which result in different transport capabilities. P-gp functionality and the effect of P-gp modulation on the pharmacokinetics of novel and established drugs can be studied in vivo by positron emission tomography (PET) using carbon-11 and fluorine-18-labeled P-gp substrates and modulators. PET may demonstrate the consequences of genetic differences on tissue pharmacokinetics. Inhibitors such as calcium-channel blockers (verapamil), cyclosporin A, ONT-093, and XR9576 can modulate the P-gp functionality. With PET the effect of P-gp modulation on the bioavailability of drugs can be investigated in humans in vivo. PET also allows the measurement of the efficacy of newly developed P-gp modulators.

  7. 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

  8. Death-Associated Protein Kinase Activity Is Regulated by Coupled Calcium/Calmodulin Binding to Two Distinct Sites.

    PubMed

    Simon, Bertrand; Huart, Anne-Sophie; Temmerman, Koen; Vahokoski, Juha; Mertens, Haydyn D T; Komadina, Dana; Hoffmann, Jan-Erik; Yumerefendi, Hayretin; Svergun, Dmitri I; Kursula, Petri; Schultz, Carsten; McCarthy, Andrew A; Hart, Darren J; Wilmanns, Matthias

    2016-06-01

    The regulation of many protein kinases by binding to calcium/calmodulin connects two principal mechanisms in signaling processes: protein phosphorylation and responses to dose- and time-dependent calcium signals. We used the calcium/calmodulin-dependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site. In DAPK2, this loop comprises a novel dimerization-regulated calcium/calmodulin-binding site, in addition to a well-established calcium/calmodulin site in the C-terminal autoregulatory domain. Unexpectedly, impairment of the basic loop interaction site completely abolishes calcium/calmodulin binding and DAPK2 activity is reduced to a residual level, indicative of coupled binding to the two sites. This contrasts with the generally accepted view that kinase calcium/calmodulin interactions are autonomous of the kinase catalytic domain. Our data establish an intricate model of multi-step kinase activation and expand our understanding of how calcium binding connects with other mechanisms involved in kinase activity regulation. PMID:27133022

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

    PubMed

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

    2008-01-01

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

  10. Comment and response document on the final remedial action plan and site design for stabilization of the inactive Uranium Mill Tailings Site at Lakeview, Oregon

    SciTech Connect

    Not Available

    1991-10-01

    This report contains comments provided by the Oregon Department of Energy and responses to these comments on the final remedial action plan for the inactive uranium mill tailings site at Lakeview, Oregon.

  11. 77 FR 22776 - Agency Information Collection Activities OMB Responses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-17

    ... From the Federal Register Online via the Government Publishing Office ENVIRONMENTAL PROTECTION AGENCY Agency Information Collection Activities OMB Responses AGENCY: Environmental Protection Agency... of information unless it displays a currently valid OMB control number. The OMB control numbers...

  12. 78 FR 68053 - Agency Information Collection Activities OMB Responses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-13

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  13. 75 FR 54626 - Agency Information Collection Activities OMB Responses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-08

    ... From the Federal Register Online via the Government Publishing Office ENVIRONMENTAL PROTECTION AGENCY Agency Information Collection Activities OMB Responses AGENCY: Environmental Protection Agency... of information unless it displays a currently valid OMB control number. The OMB control numbers...

  14. 75 FR 62383 - Agency Information Collection Activities OMB Responses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-08

    ... From the Federal Register Online via the Government Publishing Office ENVIRONMENTAL PROTECTION AGENCY Agency Information Collection Activities OMB Responses AGENCY: Environmental Protection Agency... of information unless it displays a currently valid OMB control number. The OMB control numbers...

  15. 77 FR 56200 - Agency Information Collection Activities OMB Responses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-12

    ... From the Federal Register Online via the Government Publishing Office ENVIRONMENTAL PROTECTION AGENCY Agency Information Collection Activities OMB Responses AGENCY: Environmental Protection Agency... of information unless it displays a currently valid OMB control number. The OMB control numbers...

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

    PubMed Central

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

    2015-01-01

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

  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. Site Response And Slow Basin Waves In The Sacramento/San Joaquin Delta

    NASA Astrophysics Data System (ADS)

    Fletcher, J. B.; Sell, R.

    2009-12-01

    About 1700 km of levees protect farmland and communities from inundation in the delta of the Sacramento/San Joaquin Rivers at the western edge of the Great Valley in California. These levees are made from dredged soils, such as peat, and are characterized by low shear-wave velocities (about 200m/s) and are vulnerable to breaking from major earthquakes in the greater San Francisco Bay area. We have investigated the seismic response of sites in the delta by deploying a set of broadband seismometers to record local moderate-sized earthquakes. Each of the 11 sites has a 30s broadband, 3-component seismograph digitized continuously at 100 sps/channel. In the summer of 2008 we expanded the original array, which covered a small area near Bethel Is., to cover a greater proportion of the delta, and it now extends from Tracy in the south to Bethel Is. in the northwest to Eight Mile Road in the northeast, an area of about 20 by 30 km. Several of the sites are on levees and the others are on farmland. One site is on a outcrop just west of the Clifton Court Forebay in the foothills. During the last year we have recorded nine events in the M 3 to 4 range with good signal to noise at the array. Site response was estimated with spectral ratios of S waves using Black Diamond Mine, a station in the UC Berkeley seismic network, as a reference station. Site responses at levee sites typically show large resonances in the 1-3 Hz range with amplifications greater than 10 for the Sept. 6, 2008 M4.1 Alamo event. Other sites show amplifications between 2 and 7 at various frequencies between 1 and 10 Hz. Sites within the delta show late-arriving Rayleigh waves with a period of about one second. A three-element array at the Holland Marina (spacing 180-290m) shows these waves to be traveling at about 610 m/s with a back azimuth about 20 degrees off the azimuth to the epicenter. Observations of well-developed, one-second surface waves across basins are not common, but they are similar to

  19. Report on the emergency response to the event on May 14, 1997, at the plutonuim reclamation facility, Hanford Site, Richland,Washington

    SciTech Connect

    Shoop, D.S.

    1997-08-20

    On the evening of May 14,1997, a chemical explosion Occurred at the Plutonium Reclamation Facility (PRF) in the 200 West Area(200-W) of the Hanford Site. The event warranted the declaration of an Alert emergency, activation of the Hanford Emergency Response Organization (BRO), and notification of offsite agencies. As a result of the emergency declaration, a subsequent evaluation was conducted to assess: 9 the performance of the emergency response organization o the occupational health response related to emergency activities o event notifications to offsite and environmental agencies. Additionally, the evaluation was designed to: 9 document the chronology of emergency and occupational health responses and environmental notifications connected with the explosion at the facility 0 assess the adequacy of the Hanford Site emergency preparedness activities; response readiness; and emergency management actions, occupational health, and environmental actions 0 provide an analysis of the causes of the deficiencies and weaknesses in the preparedness and response system that have been identified in the evaluation of the response a assign organizational responsibility to correct deficiencies and weaknesses a improve future performance 0 adjust elements of emergency implementing procedures and emergency preparedness activities.

  20. Pythium infection activates conserved plant defense responses in mosses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The moss Physcomitrella patens (P. patens) is a useful model to study abiotic stress responses since it is highly tolerant to drought, salt and osmotic stress. However, little is known about the defense mechanisms activated in this moss after pathogen assault. Here the induction of defense responses...

  1. 78 FR 26772 - Agency Information Collection Activities OMB Responses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-08

    ... AGENCY Agency Information Collection Activities OMB Responses AGENCY: Environmental Protection Agency.... Seq.). An agency may not conduct or sponsor, and a person is not required to respond to, a collection... Information Collection Request (ICR) Number. SUPPLEMENTARY INFORMATION: OMB Responses to Agency...

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

    PubMed Central

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

    2016-01-01

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

  3. Accumulation of heavy metals and antioxidant responses in Pinus sylvestris L. needles in polluted and non-polluted sites.

    PubMed

    Kandziora-Ciupa, Marta; Ciepał, Ryszard; Nadgórska-Socha, Aleksandra; Barczyk, Gabriela

    2016-07-01

    The purpose of this study was to determine the concentrations of heavy metals (cadmium, iron, manganese, lead and zinc) in current-year, 1-year old and 2-year old needles of Pinus sylvestris L. Trees were from three heavily polluted (immediate vicinity of zinc smelter, iron smelter and power plant) and three relatively clean sites (nature reserve, ecologically clean site and unprotected natural forest community) in southern Poland. Analysis also concerned the antioxidant response and contents of protein, proline, total glutathione, non-protein thiols and activity of guaiacol peroxidase (GPX) in the needles. Generally, in pine needles from the polluted sites, the concentrations of the metals were higher and increased with the age of needles, and in most cases, antioxidant responses also were elevated. The highest levels of Cd, Pb and Zn were found in 2-year old pine needles collected near the polluted zinc smelter (respectively: 6.15, 256.49, 393.5 mg kg(-1)), Fe in 2-year old pine needles in the vicinity of the iron smelter (206.82 mg kg(-1)) and Mn in 2-year old needles at the ecologically clean site (180.32 mg kg(-1)). Positive correlations were found between Fe, Mn and Pb and the content of proteins and NPTs, between Cd and non-protein -SH groups, and between Zn and proline levels. The activity of GPX increased under the influence of Mn, while glutathione levels tended to decrease as Mn levels rose. The data obtained show that the levels of protein and non-protein -SH groups may be useful in biological monitoring, and that these ecophysiological parameters seem to be good evidence of elevated oxidative stress caused by heavy metals. PMID:27033856

  4. Three-dimensional site response at KiK-net downhole arrays

    USGS Publications Warehouse

    Thompson, Eric M.; Tanaka, Yasuo; Baise, Laurie G.; Kayen, Robert E.

    2010-01-01

    Ground motions at two Kiban-Kyoshin Network (KiK-net) strong motion downhole array sites in Hokkaido, Japan (TKCH08 in Taiki and TKCH05 in Honbetsu) illustrate the importance of three-dimensional (3D) site effects. These sites recorded the M8.0 2003 Tokachi-Oki earthquake, with recorded accelerations above 0.4 g at both sites as well as numerous ground motions from smaller events. Weak ground motions indicate that site TKCH08 is well modeled with the assumption of plane SH waves traveling through a 1D medium (SH1D), while TKCH05 is characteristic of a poor fit to the SH1D theoretical response. We hypothesized that the misfit at TKCH05results from the heterogeneity of the subsurface. To test this hypothesis, we measured four S-wave velocity profiles in the vicinity (< 300 m) of each site with the spectral analysis of surface waves (SASW) method. This KiK-net site pair is ideal for assessing the relative importance of 3D site effects and nonlinear site effects. The linear ground motions at TKCH05 isolate the 3D site effects, as we hypothesized from the linear ground motions and confirmed with our subsequent SASW surveys. The Tokachi-Oki time history at TKCH08 isolates the effects of nonlinearity from spatial heterogeneity because the 3D effects are negligible. The Tokachi-Oki time history at TKCH05 includes both nonlinear and 3D site effects. Comparisons of the accuracy of the SH1D model predictions of these surface time histories from the downhole time histories indicates that the 3D site effects are at least as important as nonlinear effects in this case. The errors associated with the assumption of a 1D medium and 1D wave propagation will be carried into a nonlinear analysis that relies on these same assumptions. Thus, the presence of 3D effects should be ruled out prior to a 1D nonlinear analysis. The SH1D residuals show that 3D effects can be mistaken for nonlinear effects.

  5. Divergent Immune Responses to Mycobacterium avium subsp. paratuberculosis Infection Correlate with Kinome Responses at the Site of Intestinal Infection

    PubMed Central

    Määttänen, Pekka; Trost, Brett; Scruten, Erin; Potter, Andrew; Kusalik, Anthony; Griebel, Philip

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease (JD) in cattle. M. avium subsp. paratuberculosis infects the gastrointestinal tract of calves, localizing and persisting primarily in the distal ileum. A high percentage of cattle exposed to M. avium subsp. paratuberculosis do not develop JD, but the mechanisms by which they resist infection are not understood. Here, we merge an established in vivo bovine intestinal segment model for M. avium subsp. paratuberculosis infection with bovine-specific peptide kinome arrays as a first step to understanding how infection influences host kinomic responses at the site of infection. Application of peptide arrays to in vivo tissue samples represents a critical and ambitious step in using this technology to understand host-pathogen interactions. Kinome analysis was performed on intestinal samples from 4 ileal segments subdivided into 10 separate compartments (6 M. avium subsp. paratuberculosis-infected compartments and 4 intra-animal controls) using bovine-specific peptide arrays. Kinome data sets clustered into two groups, suggesting unique binary responses to M. avium subsp. paratuberculosis. Similarly, two M. avium subsp. paratuberculosis-specific immune responses, characterized by different antibody, T cell proliferation, and gamma interferon (IFN-γ) responses, were also observed. Interestingly, the kinomic groupings segregated with the immune response groupings. Pathway and gene ontology analyses revealed that differences in innate immune and interleukin signaling and particular differences in the Wnt/β-catenin pathway distinguished the kinomic groupings. Collectively, kinome analysis of tissue samples offers insight into the complex cellular responses induced by M. avium subsp. paratuberculosis in the ileum and provides a novel method to understand mechanisms that alter the balance between cell-mediated and antibody responses to M. avium subsp. paratuberculosis infection. PMID

  6. Divergent immune responses to Mycobacterium avium subsp. paratuberculosis infection correlate with kinome responses at the site of intestinal infection.

    PubMed

    Määttänen, Pekka; Trost, Brett; Scruten, Erin; Potter, Andrew; Kusalik, Anthony; Griebel, Philip; Napper, Scott

    2013-08-01

    Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease (JD) in cattle. M. avium subsp. paratuberculosis infects the gastrointestinal tract of calves, localizing and persisting primarily in the distal ileum. A high percentage of cattle exposed to M. avium subsp. paratuberculosis do not develop JD, but the mechanisms by which they resist infection are not understood. Here, we merge an established in vivo bovine intestinal segment model for M. avium subsp. paratuberculosis infection with bovine-specific peptide kinome arrays as a first step to understanding how infection influences host kinomic responses at the site of infection. Application of peptide arrays to in vivo tissue samples represents a critical and ambitious step in using this technology to understand host-pathogen interactions. Kinome analysis was performed on intestinal samples from 4 ileal segments subdivided into 10 separate compartments (6 M. avium subsp. paratuberculosis-infected compartments and 4 intra-animal controls) using bovine-specific peptide arrays. Kinome data sets clustered into two groups, suggesting unique binary responses to M. avium subsp. paratuberculosis. Similarly, two M. avium subsp. paratuberculosis-specific immune responses, characterized by different antibody, T cell proliferation, and gamma interferon (IFN-γ) responses, were also observed. Interestingly, the kinomic groupings segregated with the immune response groupings. Pathway and gene ontology analyses revealed that differences in innate immune and interleukin signaling and particular differences in the Wnt/β-catenin pathway distinguished the kinomic groupings. Collectively, kinome analysis of tissue samples offers insight into the complex cellular responses induced by M. avium subsp. paratuberculosis in the ileum and provides a novel method to understand mechanisms that alter the balance between cell-mediated and antibody responses to M. avium subsp. paratuberculosis infection. PMID

  7. An Improved Description of the Seismic Response of Sites with High Plasticity Soils, Organic Clays, and Deep Soft Soil Deposits

    NASA Astrophysics Data System (ADS)

    Carlton, Brian

    Near surface soils can greatly influence the amplitude, duration, and frequency content of ground motions. The amount of their influence depends on many factors, such as the geometry and engineering properties of the soils and underlying bedrock, as well as the earthquake source mechanism and travel path. Building codes such as the 2012 International Building Code (IBC) define six site categories for seismic design of structures, which are based on the sites defined by the National Earthquake Hazards Reduction Program (NEHRP). Site categories A, B, C, D, and E are defined by the time averaged shear wave velocity over the top 30 meters of the soil deposit. Site category F is defined as sites that include liquefiable or sensitive soils, as well as sites with more than 3 meters (10 ft) of peat or highly organic clays, more than 7.5 meters (25 ft) of soil with PI > 75, and more than 37 meters (120 ft) of soft to medium stiff clays. The IBC specifies simplified procedures to calculate design spectra for NEHRP sites A through E, and requires a site specific investigation for NEHRP F sites. However, established procedures for performing the required site specific investigations for NEHRP F sites are limited. The objective of this research is to develop a simplified procedure to estimate design spectra for non-liquefiable NEHRP F sites, specifically sites with organic soils, highly plastic soils, and deep soft soil deposits. The results from this research will directly affect US practice by developing much needed guidelines in this area. There is little empirical data on the seismic response of non-liquefiable NEHRP F sites. As a result, this study focused on generating data from site response analyses. To capture the variability of ground motions, this study selected five base case scenarios according to tectonic environments and representative cases encountered in common US practice. Suites of ground motions for each scenario were created by collecting ground motions

  8. Magnetospheric impulse response for many levels of geomagnetic activity

    NASA Technical Reports Server (NTRS)

    Bargatze, L. F.; Baker, D. N.; Hones, E. W., Jr.; Mcpherron, R. L.

    1985-01-01

    The temporal relationship between the solar wind and magnetospheric activity has been studied using 34 intervals of high time resolution IMP 8 solar wind data and the corresponding AL auroral activity index. The median values of the AL index for each interval were utilized to rank the intervals according to geomagnetic activity level. The linear prediction filtering technique was then applied to model magnetospheric response as measured by the AL index to the solar wind input function VB(s). The linear prediction filtering routine produces a filter of time-lagged response coefficients which estimates the most general linear relationship between the chosen input and output parameters of the magnetospheric system. It is found that the filters are composed of two response pulses speaking at time lags of 20 and 60 min. The amplitude of the 60-min pulse is the larger for moderate activity levels, while the 20-min pulse is the larger for strong activity levels. A possible interpretation is that the 20-min pulse represents magnetospheric activity driven directly by solar wind coupling and that the 60-min pulse represents magnetospheric activity driven by the release of energy previously stored in the magnetotail. If this interpretation is correct, the linear filtering results suggest that both the driven and the unloading models of magnetospheric response are important facets of a more comprehensive response model.

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

    PubMed

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  11. 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

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

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

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

  13. Active site coupling in PDE:PKA complexes promotes resetting of mammalian cAMP signaling.

    PubMed

    Krishnamurthy, Srinath; Moorthy, Balakrishnan Shenbaga; Xin Xiang, Lim; Xin Shan, Lim; Bharatham, Kavitha; Tulsian, Nikhil Kumar; Mihalek, Ivana; Anand, Ganesh S

    2014-09-16

    Cyclic 3'5' adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and

  14. 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.

  15. 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.

  16. A specific taurine recognition site in the rabbit brain is responsible for taurine effects on thermoregulation

    PubMed Central

    Frosini, Maria; Sesti, Casilde; Saponara, Simona; Ricci, Lorenzo; Valoti, Massimo; Palmi, Mitri; Machetti, Fabrizio; Sgaragli, Giampietro

    2003-01-01

    Taurine and GABA are recognized as endogenous cryogens. In a previous study, some structural analogues of taurine, namely 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (TAG), 2-aminoethylarsonic (AEA), 2-hydroxyethanesulfonic (ISE) and (±)cis-2-aminocyclohexane sulfonic acids (CAHS) have been shown to displace [3H]taurine binding from rabbit brain synaptic membrane preparations, without interacting either with GABA-ergic systems, nor with taurine uptake mechanism, thus behaving like direct taurinergic agents. To answer the question whether the role of taurine as an endogenous cryogen depends on the activation of GABA receptors or that of specific taurine receptor(s), taurine or the above structural analogues were injected intracerebroventricularly in conscious, restrained rabbits singularly or in combination and their effects on rectal (RT)- and ear–skin temperature and gross motor behavior (GMB) were monitored. Taurine (1.2 × 10−6–4.8 × 10−5 mol) induced a dose-related hypothermia, vasodilation at ear vascular bed and inhibition of GMB. CAHS, at the highest dose tested (4.8 × 10−5 mol) induced a taurine-like effect either on RT or GMB. On the contrary ISE, injected at the same doses of taurine, induced a dose-related hyperthermia, vasoconstriction and excitation of GMB. AEA and TAG caused a dose-related hyperthermia, but at doses higher than 1.2 × 10−7 mol caused death within 24 h after treatment. CAHS (4.8 × 10−5 mol) antagonized the hyperthermic effect induced by TAG (1.2 × 10−6 mol), AEA (1.2 × 10−8 mol) or ISE (4.8 × 10−5 mol). In conclusion, these findings may indicate the existence of a recognition site specific for taurine, responsible for its effects on thermoregulation. PMID:12788808

  17. Human bone marrow stromal cells display variable anatomic site-dependent response and recovery from irradiation

    PubMed Central

    Damek-Poprawa, Monika; Stefanik, Derek; Levin, Lawrence M.; Akintoye, Sunday O.

    2010-01-01

    Objectives Orofacial bone is commonly affected by osteoradionecrosis (ORN) during head and neck cancer radiotherapy possibly due to interactions of several factors including radiation damage to resident bone marrow stromal cells (BMSCs). Irradiation causes DNA damage, triggers p53-dependent signaling resulting in either cell-cycle arrest or apoptosis. In same individuals, disproportionately higher rapid growth of orofacial BMSCs relative to those of axial/appendicular bones suggests their response to radiation is skeletally site-specific. We hypothesized that survival and osteogenic recovery capacity of irradiated human BMSCs is site-dependent based on anatomic skeletal site of origin. Methods Early passage BMSCs from maxilla, mandible and iliac crest of four normal volunteers were exposed to 2.5 to 10 Gy gamma radiation to evaluate clonogenic survival, effects on cell cycle, DNA damage, p53-related response and in vivo osteogenic regenerative capacity. Results Orofacial bone marrow stromal cells (OF-MSCs) survived higher radiation doses and recovered quicker than iliac crest (IC-MSCs) based on clonogenic survival, proliferation and accumulation in G0G1 phase. Post-irradiation p53 level was relatively unchanged but expression of p21, a downstream effector was moderately increased in OF-MSCs. Re-establishment of in vivo bone regeneration was delayed more in irradiated IC-MSCs relative to OF-MSCs. Conclusions Effect of irradiation on human BMSCs was skeletal site-specific with OF-MSCs displaying higher radio-resistance and quicker recovery than IC-MSCs. PMID:20378097

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

    PubMed

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

    2014-12-12

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

  19. Detection of leucine-independent DNA site occupancy of the yeast Leu3p transcriptional activator in vivo.

    PubMed Central

    Kirkpatrick, C R; Schimmel, P

    1995-01-01

    The product of the Saccharomyces cerevisiae LEU3 gene, Leu3p, is a transcriptional activator which regulates leucine biosynthesis in response to intracellular levels of leucine through the biosynthetic intermediate alpha-isopropylmalate. We devised a novel assay to examine the DNA site occupancy of Leu3p under different growth conditions, using a reporter gene with internal Leu3p-binding sites. Expression of the reporter is inhibited by binding of nuclear Leu3p to these sites; inhibition is dependent on the presence of the sites in the reporter, on the integrity of the Leu3p DNA-binding domain, and, surprisingly, on the presence of a transcriptional activation domain in the inhibiting protein. By this assay, Leu3p was found to occupy its binding site under all conditions tested, including high and low levels of leucine and in the presence and absence of alpha-isopropylmalate. The localization of Leu3p to the nucleus was confirmed by immunofluorescence staining of cells expressing epitope-tagged Leu3p derivatives. We conclude that Leu3p regulates transcription in vivo without changing its intracellular localization and DNA site occupancy. PMID:7623798

  20. 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.

  1. Kinetic Evidence for the Presence of Two Postaglandin Receptor Sites Regulating the Activity of Intestinal Adenylate Cyclase Sensitive to Escherichia coli Enterotoxin

    PubMed Central

    Kantor, Harvey S.; Tao, Pearl; Kiefer, Helen Chilton

    1974-01-01

    Kinetic behavior most consistent with the presence of two independent, but simultaneously acting, regulatory effector sites for prostaglandins has been presented for adenylate cyclase (EC 4.6.1.1) of rabbit intestinal epithelial cells. One site regulates activation of the catalytic site, while the other site regulates inhibition. A synthetic prostaglandin analogue, 7-oxa-13-prostynoic acid, is recognized at both sites in a concentration-dependent manner. At concentrations of 7-oxa-13-prostynoic acid less than 45 μg/ml, activation is seen, while at higher concentrations, inhibition is seen. Different naturally occurring prostaglandins appear to be site-specific. Prostaglandin E1 gives only activation of the cyclase, while prostaglandin A1 gives only inhibition of the activated cyclase. When saturating concentrations of prostaglandin E1 are used to activate adenylate cyclase, no further activation by 7-oxa-13-prostynoic acid can be elicited, indicating that both molecules activate at the same site. The similarity of inhibition constants for both 7-oxa-13-prostynoic acid and prostaglandin A1 suggests that the mode of binding is the same for both compounds and that they probably inhibit by acting at the same site. The inhibition by 7-oxa-13-prostynoic acid and by prostaglandin A1 overrides enzyme activation produced by either Escherichia coli enterotoxin, prostaglandin E1, or sodium fluoride, suggesting that in intestinal adenylate cyclase this site is the primary regulatory site (i.e., primary allosteric effector site) for enzyme activity. These data suggest that sites exist on adenylate cyclase which would allow prostaglandins to serve as the intracellular messengers by which the cell controls its adenylate-cyclase-mediated response to extracellular stimulation, as with hormones. PMID:4208548

  2. Activated oxygen alters cerebral microvascular responses in newborn pigs

    SciTech Connect

    Leffler, C.W.; Busiia, D.W.; Armstead, W.M.; Mirro, R.; Thelin, O. )

    1990-02-26

    In piglets, cerebral ischemia/reperfusion blocks prostanoid dependent cerebral vasodilation to hypercapnia (CO{sub 2}) and hypotension but not prostanoid independent dilation to isoproterenol (Isu) or constriction to norepinephrine (NE). Ischemia/reperfusion increases activated-O{sub 2} production by piglet brains. Using cranial windows in piglets, the authors investigated the hypothesis that activated oxygen can block prostanoid dependent cerebral vasodilator responses to CO{sub 2} and hypotension without altering responses to Isu and NE. Exposure to an activated oxygen generating system of xanthine oxidase, hypoxanthine, and Fe that made about 3 times the activated-O{sub 2} on the brain surface as ischemia/reperfusion caused reversible pial arteriolar dilation. After exposure, pial arteriolar dilation was reduced to CO{sub 2} and hypotension but not to Isu. NE constrictor responses were also unaltered. H{sub 2}O{sub 2} or H{sub 2}O{sub 2} + Fe caused constriction followed by reversible dilation. After exposure, pial arteriolar dilation in response to CO{sub 2} and hypotension was not altered. However, addition of xanthine oxidase and hypoxanthine with H{sub 2}O{sub 2} and Fe totally eliminated pial arteriolar dilator responses to CO{sub 2} and hypotension but did not decrease dilation caused by Isu or constriction caused by NE. The authors conclude that activated oxygen could produce the altered prostanoid dependent pial arteriolar responses observed following ischemia in piglets.

  3. 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…

  4. A Novel Peroxisome Proliferator Response Element Modulates Hepatic Low Density Lipoprotein Receptor Gene Transcription in Response to PPARδ Activation

    PubMed Central

    Shende, Vikram R.; Singh, Amar Bahadur; Liu, Jingwen

    2016-01-01

    The hepatic expression of LDLR gene is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein 2 (SREBP2). However whether additional cis-regulatory elements contribute to LDLR transcription has not been fully explored. We investigated the function of a putative PPAR-response element (PPRE) sequence motif located at −768 to −752 bases upstream of the transcription start site of human LDLR gene in response to PPARδ activation. Promoter luciferase reporter analyses showed that treating HepG2 cells with PPARδ agonist L165041 markedly increased the activity of a full-length LDLR promoter construct (pLDLR-1192) without any effects on the shorter promoter reporter pLDLR-234 that contains only the core regulatory elements SRE-1 and SP1 sites. Importantly, mutation of the PPRE sequence greatly attenuated the induction of the full-length LDLR promoter activity by L165041 without affecting rosuvastatin mediated transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays further confirmed the binding of PPARδ to the LDLR-PPRE site. Treating HepG2 cells with L165041 elevated the mRNA and protein expressions of LDLR without affecting the LDLR mRNA decay rate. The induction of LDLR expression by PPARδ agonist was further observed in liver tissue of mice and hamsters treated with L165041. Altogether, our studies identify a novel PPRE-mediated regulatory mechanism for LDLR transcription and suggest that combined treatment of statin with PPARδ agonists may have advantageous effects on LDLR expression. PMID:26443862

  5. 76 FR 10028 - Settlement Agreement for Recovery of Past Response Costs 10,000 Havana Street Site, Commerce City...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-23

    ... AGENCY Settlement Agreement for Recovery of Past Response Costs 10,000 Havana Street Site, Commerce City... Mascarenas (Settling Party) regarding the 10,000 Havana Street Site (Site), located at 10,000 Havana Street... Street, 3rd Floor, in Denver, Colorado. DATES: Comments must be submitted on or before March 25,...

  6. Seismic characterization and dynamic site response of a municipal solid waste landfill in Bangalore, India.

    PubMed

    Anbazhagan, P; SivakumarBabu, G L; Lakshmikanthan, P; VivekAnand, K S

    2016-03-01

    Seismic design of landfills requires an understanding of the dynamic properties of municipal solid waste (MSW) and the dynamic site response of landfill waste during seismic events. The dynamic response of the Mavallipura landfill situated in Bangalore, India, is investigated using field measurements, laboratory studies and recorded ground motions from the intraplate region. The dynamic shear modulus values for the MSW were established on the basis of field measurements of shear wave velocities. Cyclic triaxial testing was performed on reconstituted MSW samples and the shear modulus reduction and damping characteristics of MSW were studied. Ten ground motions were selected based on regional seismicity and site response parameters have been obtained considering one-dimensional non-linear analysis in the DEEPSOIL program. The surface spectral response varied from 0.6 to 2 g and persisted only for a period of 1 s for most of the ground motions. The maximum peak ground acceleration (PGA) obtained was 0.5 g and the minimum and maximum amplifications are 1.35 and 4.05. Amplification of the base acceleration was observed at the top surface of the landfill underlined by a composite soil layer and bedrock for all ground motions. Dynamic seismic properties with amplification and site response parameters for MSW landfill in Bangalore, India, are presented in this paper. This study shows that MSW has less shear stiffness and more amplification due to loose filling and damping, which need to be accounted for seismic design of MSW landfills in India. PMID:26759434

  7. Conformational Disorganization within the Active Site of a Recently Evolved Organophosphate Hydrolase Limits Its Catalytic Efficiency.

    PubMed

    Mabbitt, Peter D; Correy, Galen J; Meirelles, Tamara; Fraser, Nicholas J; Coote, Michelle L; Jackson, Colin J

    2016-03-01

    The evolution of new enzymatic activity is rarely observed outside of the laboratory. In the agricultural pest Lucilia cuprina, a naturally occurring mutation (Gly137Asp) in α-esterase 7 (LcαE7) results in acquisition of organophosphate hydrolase activity and confers resistance to organophosphate insecticides. Here, we present an X-ray crystal structure of LcαE7:Gly137Asp that, along with kinetic data, suggests that Asp137 acts as a general base in the new catalytic mechanism. Unexpectedly, the conformation of Asp137 observed in the crystal structure obstructs the active site and is not catalytically productive. Molecular dynamics simulations reveal that alternative, catalytically competent conformers of Asp137 are sampled on the nanosecond time scale, although these states are less populated. Thus, although the mutation introduces the new reactive group responsible for organophosphate detoxification, the catalytic efficiency appears to be limited by conformational disorganization: the frequent sampling of low-energy nonproductive states. This result is consistent with a model of molecular evolution in which initial function-changing mutations can result in enzymes that display only a fraction of their catalytic potential due to conformational disorganization. PMID:26881849

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

    SciTech Connect

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

    2010-01-12

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

  9. 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

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

    PubMed

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

    2016-06-15

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

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

    PubMed

    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. Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and the runt domain binding sites

    NASA Technical Reports Server (NTRS)

    Winchester, S. K.; Selvamurugan, N.; D'Alonzo, R. C.; Partridge, N. C.

    2000-01-01

    Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.

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

    PubMed

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

    2015-02-23

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

  14. 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

  15. Evaluating Nonpolar Surface Area and LC/MS Response: An Application for Site Occupancy Measurements for Enzyme Intermediates in Polyketide Biosynthesis

    PubMed Central

    Randall, Shan M.; Koryakina, Irina; Williams, Gavin J.; Muddiman, David C.

    2014-01-01

    RATIONALE Site occupancy measurements using LC/MS are reported throughout the literature. However, site occupancy quantification suffers from ionization bias between modified and unmodified peptides containing the active site. In this study, we explore the MS signal as a function of nonpolar surface area (NPSA) in order to better understand this bias in electrospray response. The correlation between hydrophobicity and LC/MS response was evaluated and applied to study enzyme intermediates in polyketide synthases. METHODS Site occupancy methods were developed to study acyltransferase activity. To further evaluate these methods, several standard peptides containing one cysteine residue were modified with alkylation reagents of increasing hydrophobicity to study the MS signal as a function of nonpolar surface area. RESULTS A consistent trend in MS response was observed which is dependent on the NPSA of the analyte. An optimal NPSA zone was observed for the peptides studied. CONCLUSIONS Nonpolar surface area can be used as metric to determine relative LC/MS response for peptides and evaluate site occupancy measurements. PMID:25366398

  16. 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

  17. Small Molecule Active Site Directed Tools for Studying Human Caspases.

    PubMed

    Poreba, Marcin; Szalek, Aleksandra; Kasperkiewicz, Paulina; Rut, Wioletta; Salvesen, Guy S; Drag, Marcin

    2015-11-25

    Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases, or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes. PMID:26551511

  18. Activation of brown adipose tissue mitochondrial GDP binding sites

    SciTech Connect

    Swick, A.G.

    1987-01-01

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

  19. Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site

    PubMed Central

    Barrack, Keri L.; Tulloch, Lindsay B.; Burke, Lynsey-Ann; Fyfe, Paul K.; Hunter, William N.

    2011-01-01

    Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species, protozoa that are responsible for a range of serious diseases found in tropical and subtropical parts of the world. As part of a structure-based approach to inhibitor development, specifically targeting Leishmania species, well ordered crystals of L. donovani PTR1 were sought to support the characterization of complexes formed with inhibitors. An efficient system for recombinant protein production was prepared and the enzyme was purified and crystallized in an orthorhombic form with ammonium sulfate as the precipitant. Diffraction data were measured to 2.5 Å resolution and the structure was solved by molecular replacement. However, a sulfate occupies a phosphate-binding site used by NADPH and occludes cofactor binding. The nicotinamide moiety is a critical component of the active site and without it this part of the structure is disordered. The crystal form obtained under these conditions is therefore unsuitable for the characterization of inhibitor complexes. PMID:21206018

  20. Estimation of response-spectral values as functions of magnitude, distance, and site conditions

    USGS Publications Warehouse

    Joyner, W.B.; Boore, David M.

    1982-01-01

    We have developed empirical predictive equations for the horizontal pseudo-velocity response at 5-percent damping for 12 different periods from 0.1 to 4.0 s. Using a multiple linear-regression method similar to the one we used previously for peak horizontal acceleration and velocity, we analyzed response spectra period by period for 64 records of 12 shallow earthquakes in western North America, including the recent Coyote Lake and Imperial Valley, California, earthquakes. The resulting predictive equations show amplification of the response values at soil sites for periods greater than or equal to 0.5 s, with maximum amplification exceeding a factor of 2 at 1.5 s. For periods less than 0.5 s there is no statistically significant difference between rock sites and the soil sites represented in the data set. These results are consistent with those of several earlier studies. A particularly significant aspect of the predictive equations is that the response values at different periods are different functions of magnitude (confirming earlier results by McGuire and by Trifunac and Anderson). The slope of the least-squares straight line relating log response to moment magnitude ranges from 0.21 at a period of 0.1 s to greater than 0.5 at periods of 1 s and longer. This result indicates that the conventional practice of scaling a constant spectral shape by peak acceleration will not give accurate answers. The Newmark and Hall method of spectral scaling, using both peak acceleration and peak velocity, largely avoids this error. Comparison of our spectra with the Regulatory Guide 1.60 spectrum anchored at the same value at 0.1 s shows that the Regulatory Guide 1.60 spectrum is exceeded at soil sites for a magnitude of 7.5 at all distances for periods greater than about 0.5 s. Comparison of our spectra for soil sites with the corresponding ATC-3 curve of lateral design force coefficients for the highest seismic zone indicates that the ATC-3 curve is exceeded within about 5 km

  1. Environmental proteomics reveals early microbial community responses to biostimulation at a uranium- and nitrate-contaminated site

    SciTech Connect

    Chourey, Karuna; Nissen, Silke; Vishnivetskaya, T.; Shah, Manesh B; Pffifner, Susan; Hettich, Robert {Bob} L; Loeffler, Frank E

    2013-01-01

    High performance mass spectrometry instrumentation coupled with improved protein extraction techniques enable metaproteomics to identify active members of soil and groundwater microbial communities. Metaproteomics workflows were applied to study the initial responses (i.e., 4 days post treatment) of the indigenous aquifer microbiota to biostimulation with emulsified vegetable oil (EVO) at a uranium-contaminated site. Members of the Betaproteobacteria (i.e., Dechloromonas, Ralstonia, Rhodoferax, Polaromonas, Delftia, Chromobacterium) and Firmicutes dominated the biostimulated aquifer community. Proteome characterization revealed distinct differences in protein expression between the microbial biomass collected from groundwater influenced by biostimulation and groundwater collected up-gradient of the EVO injection points. In particular, proteins involved in ammonium assimilation, EVO degradation, and polyhydroxybutyrate (PHB) granule formation were prominent following biostimulation. Interestingly, the atypical NosZ of a Dechloromonas sp. was highly expressed suggesting active nitrous oxide (N2O) respiration. c-type cytochromes were barely detected, as was citrate synthase, a biomarker for hexavalent uranium reduction activity, suggesting that metal reduction has not commenced 4 days post EVO delivery. Environmental metaproteomics identified microbial community responses to biostimulation and elucidated active pathways demonstrating the value of this technique for complementing nucleic acid-based approaches.

  2. Site-dependent biomechanical responses of chondrocytes in the rabbit knee joint.

    PubMed

    Fick, J M; Ronkainen, A; Herzog, W; Korhonen, R K

    2015-11-26

    Biomechanical responses of chondrocytes were determined in specific locations within the superficial zone of patellar, femoral groove, femoral condyle and tibial plateau cartilages obtained from female New Zealand White rabbits. A confocal laser scanning microscope combined with a custom indentation system was utilized for experimentation. Changes in cell volumes and dimensions (i.e. cell height, width and depth) due to loading, global, local axial and transverse strains were determined for each site. Tissue composition and structure was analysed at each indentation site with digital densitometry, polarized light microscopy and Fourier transform infrared imaging spectroscopy. Patellar cells underwent greater volume decreases (compared to femoral groove cells; p<0.05) primarily due to greater decreases in cell height (p<0.05), consistent with greater levels of both global and local axial strains (p<0.05). Lateral condyle cells underwent greater volume decreases (compared to lateral plateau cells; p<0.05) primarily due to greater decreases in cell height, consistent with greater levels of tissue strains (p<0.05). Medial condyle cells underwent smaller volume decreases (compared to medial plateau cells; p<0.05) primarily due to elevated cell expansions in the depth direction, which was consistent with greater levels of minor transverse strains (p<0.05). Site-dependent differences in collagen orientation angles agreed conceptually with the observed cell dimensional changes. Chondrocyte biomechanical responses were highly site-dependent and corresponded primarily with the orientation of the collagen fibrils. The observed differences were thought to be due to the different biomechanical loading conditions at each site. PMID:26601568

  3. Participation in Peer Response as Activity: An Examination of Peer Response Stances from an Activity Theory Perspective

    ERIC Educational Resources Information Center

    Zhu, Wei; Mitchell, Deborah A.

    2012-01-01

    This article reports a case study that examined English as a Second Language students' peer response stances from an activity theory perspective. More specifically, the study was guided by the constructs of activity and motive/object in Leont'ev's theory. Multiple sources of data were collected from two native Spanish-speaking students enrolled in…

  4. 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

  5. Active urea transport by the skin of Bufo viridis: Amiloride- and phloretin-sensitive transport sites

    SciTech Connect

    Rapoport, J.; Abuful, A.; Chaimovitz, C.; Noeh, Z.; Hays, R.M. Albert Einstein College of Medicine, New York, NY )

    1988-09-01

    Urea is actively transported inwardly (J{sub i}) across the skin of the green toad Bufo viridis. J{sub i} is markedly enhanced in toads adapted to hypertonic saline. The authors studied urea transport across the skin of Bufo viridis under a variety of experimental conditions, including treatment with amiloride and phloretin, agents that inhibit urea permeability in the bladder of Bufo marinus. Amiloride (10{sup {minus}4} M) significantly inhibited J{sub i} in both adapted and unadapted animals and was unaffected by removal of sodium from the external medium. Phloretin (10{sup {minus}4} M) significantly inhibited J{sub i} in adapted animals by 23-46%; there was also a reduction in J{sub i} in unadapted toads at 10{sup {minus}4} and 5 {times} 10{sup {minus}4} M phloretin. A dose-response study revealed that the concentration of phloretin causing half-maximal inhibition (K{sub {1/2}}) was 5 {times} 10{sup {minus}4} M for adapted animals. J{sub i} was unaffected by the substitution of sucrose for Ringer solution or by ouabain. They conclude (1) the process of adaptation appears to involve an increase in the number of amiloride- and phloretin-inhibitable urea transport sites in the skin, with a possible increase in the affinity of the sites for phloretin; (2) the adapted skin resembles the Bufo marinus urinary bladder with respect to amiloride and phloretin-inhibitable sites; (3) they confirm earlier observations that J{sub i} is independent of sodium transport.

  6. Evaluating, Migrating, and Consolidating Databases and Applications for Long-Term Surveillance and Maintenance Activities at the Rocky Flats Site

    SciTech Connect

    Surovchak, S.; Marutzky, S.; Thompson, B.; Miller, K.; Labonte, E.

    2006-07-01

    The U.S. Department of Energy (DOE) Office of Legacy Management (LM) is assuming responsibilities for long-term surveillance and maintenance (LTS and M) activities at the Rocky Flats Environmental Technology Site (RFETS) during fiscal year 2006. During the transition, LM is consolidating databases and applications that support these various functions into a few applications which will streamline future management and retrieval of data. This paper discussed the process of evaluating, migrating, and consolidating these databases and applications for LTS and M activities and provides lessons learned that will benefit future transitions. (authors)

  7. Study of chaperone-like activity of human haptoglobin: conformational changes under heat shock conditions and localization of interaction sites.

    PubMed

    Ettrich, Rüdiger; Brandt, Wolfgang; Kopecký, Vladimír; Baumruk, Vladimír; Hofbauerová, Katerina; Pavlícek, Zdenek

    2002-10-01

    With respect to the mechanism of chaperone-like activity, we examined the behavior of haptoglobin under heat shock conditions. Secondary structure changes during heat treatment were followed by circular dichroism, Raman and infrared spectroscopy. A model of the haptoglobin tetramer, based on its sequence homology with serine proteases and the CCP modules, has been proposed. Sequence regions responsible for the chaperone-like activity were not fully identical with the region that takes part in formation of the hemoglobin-haptoglobin complex. We can postulate the presence of at least two different chaperone-binding sites on each haptoglobin heavy chain. PMID:12452443

  8. Ongoing activity in the optic tectum is correlated on a trial-by-trial basis with the pupil dilation response.

    PubMed

    Netser, Shai; Dutta, Arkadeb; Gutfreund, Yoram

    2014-03-01

    The selection of the appropriate stimulus to induce an orienting response is a basic task thought to be partly achieved by tectal circuitry. Here we addressed the relationship between neural activity in the optic tectum (OT) and orienting behavioral responses. We recorded multiunit activity in the intermediate/deep layers of the OT of the barn owl simultaneously with pupil dilation responses (PDR, a well-known orienting response common to birds and mammals). A trial-by-trial analysis of the responses revealed that the PDR generally did not correlate with the evoked neural responses but significantly correlated with the rate of ongoing neural activity measured shortly before the stimulus. Following this finding, we characterized ongoing activity in the OT and showed that in the intermediate/deep layers it tended to fluctuate spontaneously. It is characterized by short periods of high ongoing activity during which the probability of a PDR to an auditory stimulus inside the receptive field is increased. These high-ongoing activity periods were correlated with increase in the power of gamma band local field potential oscillations. Through dual recordings, we showed that the correlation coefficients of ongoing activity decreased as a function of distance between recording sites in the tectal map. Significant correlations were also found between recording sites in the OT and the forebrain entopallium. Our results suggest that an increase of ongoing activity in the OT reflects an internal state during which coupling between sensory stimulation and behavioral responses increases. PMID:24304859

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

    PubMed Central

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

    2015-01-01

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

  10. Multiple roles of the active site lysine of Dopa decarboxylase.

    PubMed

    Bertoldi, Mariarita; Voltattorni, Carla Borri

    2009-08-15

    The pyridoxal 5'-phosphate dependent-enzyme Dopa decarboxylase, responsible for the irreversible conversion of l-Dopa to dopamine, is an attractive drug target. The contribution of the pyridoxal-Lys303 to the catalytic mechanisms of decarboxylation and oxidative deamination is analyzed. The K303A variant binds the coenzyme with a 100-fold decreased apparent equilibrium binding affinity with respect to the wild-type enzyme. Unlike the wild-type, K303A in the presence of l-Dopa displays a parallel progress course of formation of both dopamine and 3,4-dihydroxyphenylacetaldehyde (plus ammonia) with a burst followed by a linear phase. Moreover, the finding that the catalytic efficiencies of decarboxylation and of oxidative deamination display a decrease of 1500- and 17-fold, respectively, with respect to the wild-type, is indicative of a different impact of Lys303 mutation on these reactions. Kinetic analyses reveal that Lys303 is involved in external aldimine formation and hydrolysis as well as in product release which affects the rate-determining step of decarboxylation. PMID:19580779

  11. Responsiveness of G protein-coupled odorant receptors is partially attributed to the activation mechanism

    PubMed Central

    Yu, Yiqun; de March, Claire A.; Ni, Mengjue J.; Adipietro, Kaylin A.; Golebiowski, Jérôme; Matsunami, Hiroaki; Ma, Minghong

    2015-01-01

    Mammals detect and discriminate numerous odors via a large family of G protein-coupled odorant receptors (ORs). However, little is known about the molecular and structural basis underlying OR response properties. Using site-directed mutagenesis and computational modeling, we studied ORs sharing high sequence homology but with different response properties. When tested in heterologous cells by diverse odorants, MOR256-3 responded broadly to many odorants, whereas MOR256-8 responded weakly to a few odorants. Out of 36 mutant MOR256-3 ORs, the majority altered the responses to different odorants in a similar manner and the overall response of an OR was positively correlated with its basal activity, an indication of ligand-independent receptor activation. Strikingly, a single mutation in MOR256-8 was sufficient to confer both high basal activity and broad responsiveness to this receptor. These results suggest that broad responsiveness of an OR is at least partially attributed to its activation likelihood. PMID:26627247

  12. Geological Influence on the Site Response of Bantul Earthquake at Yogyakarta Special Province, Indonesia

    NASA Astrophysics Data System (ADS)

    Karnawati, D.; Pramumijoyo, S.; Hussein, S.; Anderson, R.; Ratdomopurbo, A.

    2007-05-01

    On May 27, 2006 a magnitude 6.2 earthquake struck Yogyakarta Special Province in Central Java. The earthquake designated the Bantul earthquake resulted in the deaths of 5,800 and left 200,000 families homeless. Over 280,000 buildings were damaged or destroyed. Housing quality was generally poor with no accounting for earthquake-resistant design. Housing and building materials were also of poor quality. In spite of the poor design and construction materials, local geological conditions were determined be a significant influence on the site response which affected the intensity of building damage. In addition, strong ground shaking was unusually long at 57 seconds. In response to the findings of the initial field reconnaissance a series of micro-tremor surveys supported by aerial and satellite image interpretations, ground penetration radar and magneto telluric surveys, as well as engineering geological site investigations were conducted. The objectives of the surveys and investigations were: to investigate various factors controlling the levels of site response which induced damage to homes and buildings; and, to produce a seismic hazard micro-zonation map. Provision of this micro-zonation map is crucial to support the enhancement of building code and landuse management in the earthquake prone area at Bantul. The Bantul area is located in a valley formed a graben. This valley was due to two major normal faults extending towards North East - South West. The east border of the valley is bounded major normal fault formed the Progo River, whilst at the west border another major normal fault formed the Opak-Oya Rivers. Drilling correlations of the bedrock (of andesitic breccia) in the valley, was found at a depth of about 40 m below the existing ground surface. Bedrock was covered by a layer of clay produced from paleo-swamps with various thicknesses from 1 to 2 m. After the clay deposition, the basin then was filled by loose fluvial sediments consisting of gravelly sand

  13. 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

  14. 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.

  15. Phagocytic activity and hyperpolarizing responses in L-strain mouse fibroblasts.

    PubMed Central

    Okada, Y; Tsuchiya, W; Yada, T; Yano, J; Yawo, H

    1981-01-01

    1. Fibroblastic L cells not only respond with a slow hyperpolarizing potential change to a mechanical or electrical stimulus but also show spontaneous, repetitive hyperpolarizations (i.e. membrane potential oscillation). 2. Almost all the cells can actively take up latex beads whose surfaces were treated by U.V. irradiation. 3. Non-phagocytic L cells hardly showed hyperpolarizing responses, while hyperpolarizing responses were obtained in all the phagocytic L cells. The exposure of the cell surface to beads, however, did not trigger the generation of hyperpolarizing responses. 4. Metabolic inhibitors, low temperature and cytochalasin B inhibited both the uptake of beads and the hyperpolarizing responses. 5. Increasing the external concentration of Ca2+ induced a remarkable stimulation of the phagocytosis of beads. Mg2+ and Ba2+, which inhibited hyperpolarizing responses due to competition for Ca2+ sites on the outer surface of the membrane, significantly suppressed the uptake of beads. 6. Verapamil, a Ca2+ channel blocker, inhibited not only hyperpolarizing membrane responses but also ingestion of beads. 7. It is concluded that the Ca2+ inflow on the hyperpolarizing membrane responses is closely associated with the phagocytic activity in L cells, probably through activation of the microfilament assembly. Images Plate 1 PMID:7024506

  16. Fatal attraction: vegetation responses to nutrient inputs attract herbivores to infectious anthrax carcass sites

    PubMed Central

    Turner, Wendy C.; Kausrud, Kyrre L.; Krishnappa, Yathin S.; Cromsigt, Joris P. G. M.; Ganz, Holly H.; Mapaure, Isaac; Cloete, Claudine C.; Havarua, Zepee; Küsters, Martina; Getz, Wayne M.; Stenseth, Nils Chr.

    2014-01-01

    Parasites can shape the foraging behaviour of their hosts through cues indicating risk of infection. When cues for risk co-occur with desired traits such as forage quality, individuals face a trade-off between nutrient acquisition and parasite exposure. We evaluated how this trade-off may influence disease transmission in a 3-year experimental study of anthrax in a guild of mammalian herbivores in Etosha National Park, Namibia. At plains zebra (Equus quagga) carcass sites we assessed (i) carcass nutrient effects on soils and grasses, (ii) concentrations of Bacillus anthracis (BA) on grasses and in soils, and (iii) herbivore grazing behaviour, compared with control sites, using motion-sensing camera traps. We found that carcass-mediated nutrient pulses improved soil and vegetation, and that BA is found on grasses up to 2 years after death. Host foraging responses to carcass sites shifted from avoidance to attraction, and ultimately to no preference, with the strength and duration of these behavioural responses varying among herbivore species. Our results demonstrate that animal carcasses alter the environment and attract grazing hosts to parasite aggregations. This attraction may enhance transmission rates, suggesting that hosts are limited in their ability to trade off nutrient intake with parasite avoidance when relying on indirect cues. PMID:25274365

  17. Source and site response study of the 2008 Mount Carmel, Illinois, earthquake

    USGS Publications Warehouse

    Hartzell, S.; Mendoza, C.

    2011-01-01

    Two separate inversions are performed using the ground-motion data from the 2008 Mount Carmel, Illinois, earthquake. One uses aftershocks as empirical Green’s functions to determine a finite-fault slip distribution. The second uses mainshock ground-motion spectra to calculate source, path, and site response parameters. The slip inversion reveals a prominent asperity at the hypocenter with an area of approximately 6 km2, moment of 7.0 x 1023 dyn cm (Mw 5.20), and stress drop of about 100 bars. Considering all major and minor slip, the total moment is 1.7 x 1024 dyn cm (Mw=5.45). The rupture velocity is not resolvable due to the small source area. After fixing the geometric spreading, the source, path, and site parameter inversion yields a similar moment of 8.8 x 1023 dyn cm (Mw 5.26) and a corner frequency of 0.89 Hz, which also give a stress drop of approximately 100 bars. Our combined geometric and anelastic attenuation function, Q(f)r-b=1137f0.12r-0.94, fits the regional spectral amplitudes, where the data is more plentiful, as well as previously derived attenuation relationships. Site response spectra show prominent resonant frequencies that correlate with the thickness of Mississippi River sediments and Mississippi embayment deposits. In addition, higher frequency resonance peaks are observed that most likely represent higher mode resonances and resonances from shallower structure.

  18. 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.

  19. 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.

  20. 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...

  1. Transcriptional activation of human 12-lipoxygenase gene promoter is mediated through Sp1 consensus sites in A431 cells.

    PubMed Central

    Liu, Y W; Arakawa, T; Yamamoto, S; Chang, W C

    1997-01-01

    The functional 5' flanking region of the human 12-lipoxygenase in epidermoid carcinoma A431 cells was characterized. By a primer extension method, the transcription initiation sites were mapped at -47 adenosine, -48 guanosine and -55 guanosine upstream of the ATG translation start codon. Transient transfection with a series of 5' and 3' deletion constructs showed that the 5' flanking region spanning from -224 to -100 bp was important for the basal expression of 12-lipoxygenase gene. Gel mobility shift assays with antibodies of transcription factors showed that both Sp1 and Sp3 required highly GC-rich Sp1 sites within this region for binding. Disruption of two Sp1 recognition motifs residing at -158 to -150 bp and -123 to -114 bp by site-directed mutagenesis markedly reduced the basal 12-lipoxygenase promoter activity and abolished the retarded bands in a gel-shift assay, indicating that these two Sp1-binding sites were essential for gene expression. The same two Sp1-binding sites in this promoter region were also responsible for epidermal growth factor (EGF)-induced expression of 12-lipoxygenase gene. Moreover, EGF also induced the transcriptional activation of luciferase driven by SV40 early promoter, which contained rich Sp1-binding sites. Taken together, the results suggest that two specific Sp1 consensus sites are involved in the mediation of the basal promoter activity as well as EGF induction of the 12-lipoxygenase gene and that Sp1 and Sp3 transcription factors might have a role in their regulation. PMID:9164849

  2. Investigation of Nonlinear Site Response and Seismic Compression from Case History Analysis and Laboratory Testing

    NASA Astrophysics Data System (ADS)

    Yee, Eric

    In this thesis I address a series of issues related to ground failure and ground motions during earthquakes. A major component is the evaluation of cyclic volumetric strain behavior of unsaturated soils, more commonly known as seismic compression, from advanced laboratory testing. Another major component is the application of nonlinear and equivalent linear ground response analyses to large-strain problems involving highly nonlinear dynamic soil behavior. These two components are merged in the analysis of a truly unique and crucial field case history of nonlinear site response and seismic compression. My first topic concerns dynamic soil testing for relatively small strain dynamic soil properties such as threshold strains, gammatv. Such testing is often conducted using specialized devices such as dual-specimen simple-shear, as devices configured for large strain testing produce noisy signals in the small strain range. Working with a simple shear device originally developed for large-strain testing, I extend its low-strain capabilities by characterizing noisy signals and utilizing several statistical methods to extract meaningful responses in the small strain range. I utilize linear regression of a transformed variable to estimate the cyclic shear strain from a noisy signal and the confidence interval on its amplitude. I utilize Kernel regression with the Nadaraya-Watson estimator and a Gaussian kernel to evaluate vertical strain response. A practical utilization of these techniques is illustrated by evaluating threshold shear strains for volume change with a procedure that takes into account uncertainties in the measured shear and vertical strains. My second topic concerns the seismic compression characteristics of non-plastic and low-plasticity silty sands with varying fines content (10 ≤ FC ≤ 60%). Simple shear testing was performed on various sand-fines mixtures at a range of modified Proctor relative compaction levels ( RC) and degrees-of-saturation (S

  3. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    SciTech Connect

    Not Available

    1994-02-01

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

  4. 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

  5. 78 FR 8190 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-05

    ...BOEM is reopening the comment period announced in the Notice of Intent to Prepare an Environmental Assessment (EA) for Commercial Wind Leasing and Site Assessment Activities on the OCS Offshore North...

  6. Selectivity loss of Pt/CeO{sub 2} PROX catalysts at low CO concentrations: mechanism and active site study.

    SciTech Connect

    Polster, C. S.; Zhang, R.; Cyb, M. T.; Miller, J. T.; Baertsch, C. D.

    2010-07-01

    CO and H{sub 2} oxidation were studied over a series of Pt/CeO{sub 2} catalysts with differing Pt loadings and dispersions. Kinetic rate analysis confirms the presence of dual Langmuir-Hinshelwood (L-H) and Mars and van Krevelen (M-vK) pathways and is used to explain the loss in CO oxidation selectivity at low CO concentrations. In situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) shows the strong CO coverage dependence on both CO and O{sub 2} concentrations and explains the transition from L-H to M-vK reaction character. Redox site measurements are performed on Pt/CeO{sub 2} catalysts by anaerobic titrations under conditions where the M-vK pathway dominates the reaction rate. Similar redox site densities per interfacial Pt atom suggest that interfacial Pt-O-Ce sites are responsible for M-vK redox activity.

  7. Wetland survey of selected areas in the K-24 Site Area of responsibility

    SciTech Connect

    Rosensteel, B.A.; Awl, D.J.

    1995-07-01

    In accordance with DOE Regulations for Compliance with Floodplain/Wetlands Environmental Review Requirements, wetland surveys were conducted in selected areas within the K-25 Area of Responsibility during the summer of 1994. These areas are Mitchell Branch, Poplar Creek, the K-770 OU, Duct Island Peninsula, the Powerhouse area, and the K-25 South Corner. Previously surveyed areas included in this report are the main plant area of the K-25 Site, the K-901 OU, the AVLIS site, and the K-25 South Site. Wetland determinations were based on the USACE methodology. Forty-four separate wetland areas, ranging in size from 0.13 to 4.23 ha, were identified. Wetlands were identified in all of the areas surveyed with the exception of the interior of the Duct Island Peninsula and the main plant area of the K-25 Site. Wetlands perform functions such as floodflow alteration, sediment stabilization, sediment and toxicant retention, nutrient transformation, production export, and support of aquatic species and wildlife diversity and abundance. The forested, scrub-shrub, and emergent wetlands identified in the K-25 area perform some or all of these functions to varying degrees.

  8. Baseline Brain Activity Predicts Response to Neuromodulatory Pain Treatment

    PubMed Central

    Jensen, Mark P.; Sherlin, Leslie H.; Fregni, Felipe; Gianas, Ann; Howe, Jon D.; Hakimian, Shahin

    2015-01-01

    Objectives The objective of this study was to examine the associations between baseline electroencephalogram (EEG)-assessed brain oscillations and subsequent response to four neuromodulatory treatments. Based on available research, we hypothesized that baseline theta oscillations would prospectively predict response to hypnotic analgesia. Analyses involving other oscillations and the other treatments (meditation, neurofeedback, and both active and sham transcranial direct current stimulation) were viewed as exploratory, given the lack of previous research examining brain oscillations as predictors of response to these other treatments. Design Randomized controlled study of single sessions of four neuromodulatory pain treatments and a control procedure. Methods Thirty individuals with spinal cord injury and chronic pain had their EEG recorded before each session of four active treatments (hypnosis, meditation, EEG biofeedback, transcranial direct current stimulation) and a control procedure (sham transcranial direct stimulation). Results As hypothesized, more presession theta power was associated with greater response to hypnotic analgesia. In exploratory analyses, we found that less baseline alpha power predicted pain reduction with meditation. Conclusions The findings support the idea that different patients respond to different pain treatments and that between-person treatment response differences are related to brain states as measured by EEG. The results have implications for the possibility of enhancing pain treatment response by either 1) better patient/treatment matching or 2) influencing brain activity before treatment is initiated in order to prepare patients to respond. Research is needed to replicate and confirm the findings in additional samples of individuals with chronic pain. PMID:25287554

  9. Inducing in situ, nonlinear soil response applying an active source

    USGS Publications Warehouse

    Johnson, P.A.; Bodin, P.; Gomberg, J.; Pearce, F.; Lawrence, Z.; Menq, F.-Y.

    2009-01-01

    [1] It is well known that soil sites have a profound effect on ground motion during large earthquakes. The complex structure of soil deposits and the highly nonlinear constitutive behavior of soils largely control nonlinear site response at soil sites. Measurements of nonlinear soil response under natural conditions are critical to advancing our understanding of soil behavior during earthquakes. Many factors limit the use of earthquake observations to estimate nonlinear site response such that quantitative characterization of nonlinear behavior relies almost exclusively on laboratory experiments and modeling of wave propagation. Here we introduce a new method for in situ characterization of the nonlinear behavior of a natural soil formation using measurements obtained immediately adjacent to a large vibrator source. To our knowledge, we are the first group to propose and test such an approach. Employing a large, surface vibrator as a source, we measure the nonlinear behavior of the soil by incrementally increasing the source amplitude over a range of frequencies and monitoring changes in the output spectra. We apply a homodyne algorithm for measuring spectral amplitudes, which provides robust signal-to-noise ratios at the frequencies of interest. Spectral ratios are computed between the receivers and the source as well as receiver pairs located in an array adjacent to the source, providing the means to separate source and near-source nonlinearity from pervasive nonlinearity in the soil column. We find clear evidence of nonlinearity in significant decreases in the frequency of peak spectral ratios, corresponding to material softening with amplitude, observed across the array as the source amplitude is increased. The observed peak shifts are consistent with laboratory measurements of soil nonlinearity. Our results provide constraints for future numerical modeling studies of strong ground motion during earthquakes.

  10. Patterning of sympathetic nerve activity in response to vestibular stimulation

    NASA Technical Reports Server (NTRS)

    Kerman, I. A.; McAllen, R. M.; Yates, B. J.

    2000-01-01

    Growing evidence suggests a role for the vestibular system in regulation of autonomic outflow during postural adjustments. In the present paper we review evidence for the patterning of sympathetic nerve activity elicited by vestibular stimulation. In response to electrical activation of vestibular afferents, firing of sympathetic nerves located throughout the body is altered. However, activity of the renal nerve is most sensitive to vestibular inputs. In contrast, high-intensity simultaneous activation of cutaneous and muscle inputs elicits equivalent changes in firing of the renal, superior mesenteric and lumbar colonic nerves. Responses of muscle vasoconstrictor (MVC) efferents to vestibular stimulation are either inhibitory (Type I) or are comprised of a combination of excitation and inhibition (Type II). Interestingly, single MVC units located in the hindlimb exhibited predominantly Type I responses while those located in the forelimb and face exhibited Type II responses. Furthermore, brachial and femoral arterial blood flows were dissociated in response to vestibular stimulation, such that brachial vascular resistance increased while femoral resistance decreased. These studies demonstrate that vestibulosympathetic reflexes are patterned according to both the anatomical location and innervation target of a particular sympathetic nerve, and can lead to distinct changes in local blood flow.

  11. Prefrontal Cortex Activity Related to Abstract Response Strategies

    PubMed Central

    Genovesio, Aldo; Brasted, Peter J.; Mitz, Andrew R.; Wise, Steven P.

    2005-01-01

    Overview In monkeys, foraging strategies depend not only on a context established by spatial or symbolic cues, but also on the relations among cues. Genovesio et al. recorded the activity of prefrontal cortex neurons while monkeys chose a strategy based on the relation between consecutive symbolic cues. For the same cues and actions, the monkeys also learned fixed responses to the same symbols. Many neurons had activity selective for a given strategy, others for whether the monkeys’ response choice depended on a symbol or the relation between symbols. These findings indicate that the primate prefrontal cortex contributes to implementing abstract strategies. PMID:16039571

  12. EMERGENCY RESPONSE EQUIPMENT TO CLEAN UP HAZARDOUS CHEMICAL RELEASES AT SPILLS AND UNCONTROLLED WASTE SITES

    EPA Science Inventory

    This paper reviews some of the research activities of the U.S. Environmental Protection Agency (EPA) regarding the development of emergency response equipment to control hazardous chemical releases. Several devices and systems have been developed by EPA for environmental emergenc...

  13. 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

  14. Source spectra and site-response estimates using the Coda of Lg waves in western Europe

    NASA Astrophysics Data System (ADS)

    Chevrot, S.; Cansi, Y.

    Source spectra are investigated by using regional events recorded by the LDG French short-period seismic network. Empirical relations describing the Lg coda envelope allow us to separate source, site and propagation effects. 28 events recorded by 14 stations of the LDG network are selected for this study. Source spectra and site-response are determined in the frequency-band of 1-16 Hz. A Lg-coda magnitude can be defined from these source spectra. Magnitudes obtained by this technique are strongly correlated to local magnitudes given by the LDG bulletin but the standard deviation is reduced by a factor 3. Other parameters such as siteresponse, corner-frequency and coda Q are determined very accurately.

  15. 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

  16. Hazard Ranking System evaluation of CERCLA (Comprehensive Environmental Response, Compensation, and Liability Act) inactive waste sites at Hanford: Volume 1, Evaluation methods and results

    SciTech Connect

    Stenner, R.D.; Cramer, K.H.; Higley, K.A.; Jette, S.J.; Lamar, D.A.; McLaughlin, T.J.; Sherwood, D.R.; Van Houten, N.C.

    1988-10-01

    The purpose of this report is to formally document the individual site Hazard Ranking System (HRS) evaluations conducted as part of the preliminary assessment/site inspection (PA/SI) activities at the US Department of Energy (DOE) Hanford Site. These activities were carried out pursuant to the DOE orders that describe the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) Program addressing the cleanup of inactive waste sites. These orders incorporate the US Environmental Protection Agency methodology, which is based on the Superfund Amendments and Reauthorization Act of 1986 (SARA). The methodology includes six parts: PA/SI, remedial investigation/feasibility study, record of decision, design and implementation of remedial action, operation and monitoring, and verification monitoring. Volume 1 of this report discusses the CERCLA inactive waste-site evaluation process, assumptions, and results of the HRS methodology employed. Volume 2 presents the data on the individual CERCLA engineered-facility sites at Hanford, as contained in the Hanford Inactive Site Surveillance (HISS) Data Base. Volume 3 presents the data on the individual CERCLA unplanned-release sites at Hanford, as contained in the HISS Data Base. 34 refs., 43 figs., 47 tabs.

  17. Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

    PubMed Central

    Seibold, Steve A.; Singh, Badri Nath; Zhang, Chunfen; Kireeva, Maria; Domecq, Céline; Bouchard, Annie; Nazione, Anthony M.; Feig, Michael; Cukier, Robert I.; Coulombe, Benoit; Kashlev, Mikhail; Hampsey, Michael; Burton, Zachary F.

    2010-01-01

    Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β’ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed. PMID:20478425

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

    PubMed

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

    2014-04-01

    The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins. PMID:24635441

  19. Predominant-period site classification for response spectra prediction equations in Italy

    USGS Publications Warehouse

    Di Alessandro, Carola; Bonilla, Luis Fabian; Boore, David M.; Rovelli, Antonio; Scotti, Oona

    2012-01-01

    We propose a site‐classification scheme based on the predominant period of the site, as determined from the average horizontal‐to‐vertical (H/V) spectral ratios of ground motion. Our scheme extends Zhao et al. (2006) classifications by adding two classes, the most important of which is defined by flat H/V ratios with amplitudes less than 2. The proposed classification is investigated by using 5%‐damped response spectra from Italian earthquake records. We select a dataset of 602 three‐component analog and digital recordings from 120 earthquakes recorded at 214 seismic stations within a hypocentral distance of 200 km. Selected events are in the moment‐magnitude range 4.0≤Mw≤6.8 and focal depths from a few kilometers to 46 km. We computed H/V ratios for these data and used them to classify each site into one of six classes. We then investigate the impact of this classification scheme on empirical ground‐motion prediction equations (GMPEs) by comparing its performance with that of the conventional rock/soil classification. Although the adopted approach results in only a small reduction of the overall standard deviation, the use of H/V spectral ratios in site classification does capture the signature of sites with flat frequency‐response, as well as deep and shallow‐soil profiles, characterized by long‐ and short‐period resonance, respectively; in addition, the classification scheme is relatively quick and inexpensive, which is an advantage over schemes based on measurements of shear‐wave velocity.

  20. Specific alterations in complement protein activity of little brown myotis (Myotis lucifugus) hibernating in white-nose syndrome affected sites.

    PubMed

    Moore, Marianne S; Reichard, Jonathan D; Murtha, Timothy D; Zahedi, Bita; Fallier, Renee M; Kunz, Thomas H

    2011-01-01

    White-nose syndrome (WNS) is the most devastating condition ever reported for hibernating bats, causing widespread mortality in the northeastern United States. The syndrome is characterized by cutaneous lesions caused by a recently identified psychrophilic and keratinophylic fungus (Geomyces destructans), depleted fat reserves, atypical behavior, and damage to wings; however, the proximate cause of mortality is still uncertain. To assess relative levels of immunocompetence in bats hibernating in WNS-affected sites compared with levels in unaffected bats, we describe blood plasma complement protein activity in hibernating little brown myotis (Myotis lucifugus) based on microbicidal competence assays using Escherichia coli, Staphylococcus aureus and Candida albicans. Blood plasma from bats collected during mid-hibernation at WNS-affected sites had higher bactericidal ability against E. coli and S. aureus, but lower fungicidal ability against C. albicans when compared with blood plasma from bats collected at unaffected sites. Within affected sites during mid-hibernation, we observed no difference in microbicidal ability between bats displaying obvious fungal infections compared to those without. Bactericidal ability against E. coli decreased significantly as hibernation progressed in bats collected from an affected site. Bactericidal ability against E. coli and fungicidal ability against C. albicans were positively correlated with body mass index (BMI) during late hibernation. We also compared complement activity against the three microbes within individuals and found that the ability of blood plasma from hibernating M. lucifugus to lyse microbial cells differed as follows: E. coli>S. aureus>C. albicans. Overall, bats affected by WNS experience both relatively elevated and reduced innate immune responses depending on the microbe tested, although the cause of observed immunological changes remains unknown. Additionally, considerable trade-offs may exist between energy

  1. Planktic foraminiferal response to the Latest Danian Event in the Pacific Ocean (ODP Site 1210)

    NASA Astrophysics Data System (ADS)

    Jehle, Sofie; Bornemann, André; Deprez, Arne; Speijer, Robert

    2015-04-01

    During the Paleocene the marine ecosystem was disturbed by several transient climate events, e.g. the Dan-C2 (65.2 Ma), the Latest Danian Event (LDE, 61.75 Ma), and most known, the Paleocene-Eocene Thermal Maximum (PETM, 56 Ma). So far the LDE (or "Top Chron 27n Event") has rarely been studied in deep-sea sites with respect to the evolution and the paleoecology of planktic foraminifera. The event has already been described from Zumaia/Spain, Bjala/Bulgaria, Egypt, Shatsky Rise and Walvis Ridge. In the deep-sea the LDE is usually characterized by two distinctive Fe peaks in XRF core scanning data, paralleled by a prominent (~0.7 ‰) negative δ13C excursion (CIE) in benthic foraminifera (Westerhold et al., 2008, 2011). Benthic foraminiferal δ18O data from nearby ODP Site 1209 suggest a bottom-water temperature rise of ~2°C accompanying the negative CIE. Thus, the LDE has been considered as a further potential Paleocene "hyperthermal". Here we present data from ODP Site 1210 of the biotic response (planktic foraminifera assemblages), carbonate preservation as well as δ18O and δ13C isotope signals of the surface, subsurface and benthic taxa covering a time span of about 900 kyr around the LDE. Among others, it is mainly investigated to what extent the LDE influences the ocean ecology and especially the surface waters. Trends of both δ18O and δ13C of planktic and benthic foraminifera show negative shifts at the onset of the LDE. A 0.6‰ drop within 100 ky in planktic δ18O data suggest a temperature rise of ~2.5°C, whereas benthic foraminifera bottom water temperatures confirm a ~2°C rise like measured at Site 1209. δ13C variation is more abrupt and pronounced than the δ18O shift at the base of the LDE. The decreasing δ13C gradient between surface and subsurface dwelling foraminifera suggests a weaker and or shallower thermocline. Thermocline dwelling asymbiotic Parasubbotina rise in abundance simultaneously to the decrease in the δ13C gradient which may

  2. Active sites for NO reduction over Fe-ZSM-5 catalysts.

    PubMed

    Schwidder, M; Santhosh Kumar, M; Brückner, A; Grünert, W

    2005-02-14

    A study of Fe-ZSM-5 catalysts with variable amounts of isolated, oligomeric and heavily aggregated Fe3+ oxo sites (as evidenced by UV-Vis and EPR spectroscopic data) and their catalytic properties in the selective catalytic reduction of NO by isobutane or by NH3 is presented, which allows development of a unified concept of the active Fe sites in these reactions, according to which isolated Fe sites catalyse both SCR reactions while oligomeric sites, though also involved in the selective reduction path, limit the catalyst performance by causing the total oxidation of the reductant. PMID:15685345

  3. Laser Microdissection of Grapevine Leaves Reveals Site-Specific Regulation of Transcriptional Response to Plasmopara viticola.

    PubMed

    Lenzi, Luisa; Caruso, Carla; Bianchedi, Pier Luigi; Pertot, Ilaria; Perazzolli, Michele

    2016-01-01

    Grapevine is one of the most important fruit crops in the world, and it is highly susceptible to downy mildew caused by the biotrophic oomycete Plasmopara viticola. Gene expression profiling has been used extensively to investigate the regulation processes of grapevine-P. viticola interaction, but all studies to date have involved the use of whole leaves. However, only a small fraction of host cells is in contact with the pathogen, so highly localized transcriptional changes of infected cells may be masked by the large portion of non-infected cells when analyzing the whole leaf. In order to understand the transcriptional regulation of the plant reaction at the sites of pathogen infection, we optimized a laser microdissection protocol and analyzed the transcriptional changes in stomata cells and surrounding areas of grapevine leaves at early stages of P. viticola infection. The results indicate that the expression levels of seven P. viticola-responsive genes were greater in microdissected cells than in whole leaves, highlighting the site-specific transcriptional regulation of the host response. The gene modulation was restricted to the stomata cells and to the surrounding areas of infected tissues, indicating that the host response is mainly located at the infection sites and that short-distance signals are implicated. In addition, due to the high sensitivity of the laser microdissection technique, significant modulations of three genes that were completely masked in the whole tissue analysis were detected. The protocol validated in this study could greatly increase the sensitivity of further transcriptomic studies of the grapevine-P. viticola interaction. PMID:26546320

  4. Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase

    PubMed Central

    2015-01-01

    The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity. PMID:24517311

  5. TFEB and TFE3 cooperate in the regulation of the innate immune response in activated macrophages.

    PubMed

    Pastore, Nunzia; Brady, Owen A; Diab, Heba I; Martina, José A; Sun, Lu; Huynh, Tuong; Lim, Jeong-A; Zare, Hossein; Raben, Nina; Ballabio, Andrea; Puertollano, Rosa

    2016-08-01

    The activation of transcription factors is critical to ensure an effective defense against pathogens. In this study we identify a critical and complementary role of the transcription factors TFEB and TFE3 in innate immune response. By using a combination of chromatin immunoprecipitation, CRISPR-Cas9-mediated genome-editing technology, and in vivo models, we determined that TFEB and TFE3 collaborate with each other in activated macrophages and microglia to promote efficient autophagy induction, increased lysosomal biogenesis, and transcriptional upregulation of numerous proinflammatory cytokines. Furthermore, secretion of key mediators of the inflammatory response (CSF2, IL1B, IL2, and IL27), macrophage differentiation (CSF1), and macrophage infiltration and migration to sites of inflammation (CCL2) was significantly reduced in TFEB and TFE3 deficient cells. These new insights provide us with a deeper understanding of the transcriptional regulation of the innate immune response. PMID:27171064

  6. 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.

  7. Anabolic Responses of an Adult Cancellous Bone Site to Prostaglandin E2 in the Rat

    NASA Technical Reports Server (NTRS)

    Ito, Hiroshi; Ke, Hua Zhu; Jee, Webster S. S.; Sakou, Takashi

    1993-01-01

    The objects of this study were to determine: (1) the response of a non-growing cancellous bone site to daily prostaglandin E2 (PGE2) administration; and (2) the differences in the effects of daily PGE2, administration in growing (proximal tibial metaphysis, PTM) and non-growing cancellous bone sites (distal tibial metaphysis, DTM). Seven-month-old male Sprague-Dawley rats were given daily subcutaneous injections of 0, 1, 3 and 6 mg PGE2/kg per day for 60, 120 and 180 days. The static and dynamic histomorphometric analyses were performed on double-fluorescent labeled undecalcified distal tibial metaphyses (DTM). No age-related changes were found in static and dynamic histomorphometry of DTM cancellous bone between 7 and 13 months of age. The DTM of 7-month-old (basal controls) rats consisted of a 24.5 +/- 7.61%-metaphyseal cancellous bone mass, and a thick trabeculae (92 +/- 12 micro-m). It also had a very low tissue-base bone formation rate (3.0 +/- 7.31%/year). Exogenous PGE2 administration produced the following transient changes in a dose-response manner between zero and 60 days: (1) increased trabecular bone mass and improved architecture (increased trabecular bone area, width and number, and decreased trabecular separation); (2) increased trabecular interconnections: (3) increased bone formation parameters; and (4) decreased eroded perimeter. A new steady state with more cancellous bone mass and higher bone turnover was observed from day 60 onward, The elevated bone mass induced by the first 60 days of PGE2 treatment was maintained by another 60 and 120 days with continuous daily PGE2 treatment. When these findings were compared to those previously reported for the PTM, we found that the DTM was much more responsive to PGE2 treatment than the PTM. Percent trabecular bone area and tissue based bone formation rate increased significantly more in DTM as compared to PTM after the 60 days of 6 mg PGE2 treatment. These observations indicate that a non

  8. Computation of linear and nonlinear site response for near field ground motion

    NASA Astrophysics Data System (ADS)

    Bonilla, Luis Fabian

    The near-surface geological site conditions in the upper tens of meters are one of the dominant factors in controlling the amplitude and variation of strong ground motion during large earthquakes. The understanding of these site effects comes primarily from surface recordings. For instance, different methods to estimate site response and their variability are studied using aftershock data for the 17 January 1994 M6.7 Northridge, California earthquake. A second approach corresponds to borehole measurements. We use the Garner Valley Downhole Array (GVDA), which consists of a set of seven downhole strong-motion instruments ranging from 0 to 500 meters depth, to study site response effects. The GVDA velocity structure is first studied, then the H/ V is evaluated, and finally some considerations of 2D and 3D basin effects are also shown. These previous studies considered small to moderate earthquakes, where strain levels are small enough, so that linear wave propagation is assumed. However, for strong motions produced during large earthquakes, the soils behave nonlinearly. In this study we present evidence that nonlinearity can be directly observed in acceleration time histories such as Wildlife Refuge, 1987 Superstition Hills, CA; Kushiro Port station, 1993 Kushiro-Oki, Japan; among others. To understand the nature of nonlinear soil dynamics, we developed a model that includes anelastic dissipation of energy due to hysteresis. The hysteresis is described by the generalized Masing rules. This new hysteresis formulation, based on the classical Masing rules, has a functional representation, and depends only on one parameter that can be related to damping ratio tests. The coupling with pore pressure generation shows the degradation of the shear modulus and the yield stress during the cyclic response of the material. The simulations show amplitude reduction as well as the shift of the fundamental frequency to lower frequencies as observed on vertical arrays. The synthetic

  9. Identification of active-site residues in protease 3C of hepatitis A virus by site-directed mutagenesis.

    PubMed Central

    Gosert, R; Dollenmaier, G; Weitz, M

    1997-01-01

    Picornavirus 3C proteases (3Cpro) are cysteine proteases related by amino acid sequence to trypsin-like serine proteases. Comparisons of 3Cpro of hepatitis A virus (HAV) to those of other picornaviruses have resulted in prediction of active-site residues: histidine at position 44 (H44), aspartic acid (D98), and cysteine (C172). To test whether these residues are key members of a putative catalytic triad, oligonucleotide-directed mutagenesis was targeted to 3Cpro in the context of natural polypeptide precursor P3. Autocatalytic processing of the polyprotein containing wild-type or variant 3Cpro was tested by in vivo expression of vaccinia virus-HAV chimeras in an animal cell-T7 hybrid system and by in vitro translation of corresponding RNAs. Comparison with proteins present in HAV-infected cells showed that both expression systems mimicked authentic polyprotein processing. Individual substitutions of H44 by tyrosine and of C172 by glycine or serine resulted in complete loss of the virus-specific proteolytic cascade. In contrast, a P3 polyprotein in which D98 was substituted by asparagine underwent only slightly delayed processing, while an additional substitution of valine (V47) by glycine within putative protein 3A caused a more pronounced loss of processing. Therefore, apparently H44 and C172 are active-site constituents whereas D98 is not. The results, furthermore, suggest that substitution of amino acid residues distant from polyprotein cleavage sites may reduce proteolytic activity, presumably by altering substrate conformation. PMID:9060667

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

    PubMed Central

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

    2012-01-01

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site. PMID:21926991

  11. 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.

  12. Site response and attenuation analyses using strong motion and short-period data

    NASA Astrophysics Data System (ADS)

    Zhang, Fangyin

    Site amplification and seismic attenuation are analyzed in this study using strong ground motion records and short period records. Site amplification factors at instrumented free-field sites are inferred from recorded motions using: (1) Generalized Inversion of S-waves; (2) the H/V method (i.e., the ratio of the spectral amplitudes of the horizontal and vertical components of motion); (3) Coda-wave Inversion; (4) the Standard Spectral Ratio method; and (5) the Coda Normalization method. The site amplification factors inferred from the above methods are compared to each other. The amplification factors obtained from the Generalized Inversion are validated by the other four methods. The correlation of site amplification to site condition is also analyzed and the results demonstrate that site amplification appears to be stronger for the geologically younger (and thus softer) sediments, and the site classification in conformance with UBC provisions provides smaller scatter than the classification based on geological formation. The site amplification factors for the different site classes of the NEHRP classification (i.e., site class C, D and E), which is important for engineering application, are provided and compared with the values proposed by Borcherdt (1994) and adopted in the 1997 UBC and 2003 IBC design codes. The agreement between the site amplification obtained in this study and those proposed by Borcherdt (1994) and adopted by the design codes is good. The nonlinearity of site amplification, resulting from the intensity of ground motion, is clearly identified at selected station where this is feasible. The intrinsic and scattering attenuation are analyzed at stations SML and TYC using the Multiple Lapse Time Window (MLTW) method. The total attenuation is derived from the intrinsic and scattering attenuation. The coda attenuation is analyzed for 14 stations from coda decay curves using linear regression. The S-wave attenuation is analyzed using the coda

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

    PubMed Central

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

    2010-01-01

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

  14. Site response of heterogeneous natural deposits to harmonic excitation applied to more than 100 case histories

    NASA Astrophysics Data System (ADS)

    Chenari, Reza Jamshidi; Bostani Taleshani, Shirin Aminzadeh

    2016-06-01

    Variation of shear-wave propagation velocity (SWV) with depth was studied by analyzing more than one hundred actual SWV profiles. Linear, power, and hyperbolic variation schemes were investigated to find the most representative form for naturally occurred alluvial deposits. It was found that hyperbolic (asymptotic) variation dominates the majority of cases and it can be reliably implemented in analytical or analytical-numerical procedures. Site response analyses for a one-layer heterogeneous stratum were conducted to find an equivalent homogeneous alternative which simplifies the analysis procedure but does not compromise the accuracy of the resonance and amplification responses. Harmonic average, arithmetic average and mid-value equivalents are chosen from the literature for investigation. Furthermore, full and partial depth averaging schemes were evaluated and compared in order to verify the validity of current practices which rely upon averaging shallow depths, viz., the first 30 m of the strata. Engineering bedrock concept was discussed and the results were compared.

  15. Different cellular responses of dexmedetomidine at infected site and peripheral blood of emdotoxemic BALB/c mice.

    PubMed

    Wu, Rick Sai-Chuen; Wu, King-Chuen; Huang, Chiu-Chen; Chiang, Yi-Ying; Chen, Chia-Chen; Liao, Ching-Lung; Chu, Chin-Nan; Chung, Jing-Gung

    2015-12-01

    Various sedative agents, including dexmedetomidine (dex), induce immunosuppression, and enhance infection progression. However, there was no information on how anesthetic affects local and systemic cellular immune function. We conducted this study to examine the impact of dex on the differentiation and function of immune cells at site of inflammation and in peripheral blood during endotoxemia of mice. In BALB/c mice with and without endotoxemia, we evaluated the influence of two dosages of 5 and 50 mcg/kg/h intravenous dex on immune cells: including number of T cells (CD3), B cells (CD19), natural killer cells (CD8a), monocytes (CD11b), and macrophages (Mac-3) in peripheral blood, the activities of macrophages in peripheral blood and in peritoneal lavage, and proliferation of B and T cells and of natural killer cells activity in the spleen. Endotoxemia increased the number of CD3 T cells, CD 19 B cells and macrophages in the peripheral blood, augmented macrophage activity in the peritoneum, and increased T cell proliferation and natural killer cell activity in the spleen. Further administration of 5 mcg/kg/h dex attenuated systemic increase in number of T cells, B cells, and macrophages during endotoxemia and 50 mcg/kg/h dex significantly attenuated the increase in activity of macrophages in the peripheral blood during endotoxemia. In the peritoneum, however, 5 mcg/kg/h dex preserved and 50 mcg/kg/h dexmedetomidine enhanced the activity of macrophages during endotoxemia. Increased in proliferation of T cells in spleen during endotoxemia was attenuated by both doses of dex. Last, 50 mcg/kg/h dex enhanced natural killer cells activity during endotoxemia. While preserving the effects of endotoxemia on macrophage's activity in the infection site and natural killer cell's activity in the spleen, dex decreased systemic fulminant immune reaction in endotoxemia, by attenuating the augmented response in the number of T cells, B cells and macrophages, activity of macrophages

  16. 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

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

    SciTech Connect

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

    2007-01-01

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

  18. Assessment of the site of ventricular activation by Fourier analysis of gated blood-pool studies

    SciTech Connect

    Links, J.M.; Raichlen, J.S.; Wagner, H.N. Jr.; Reid, P.R.

    1985-01-01

    The authors studied the use of first-harmonic Fourier analysis of gated blood-pool images to assess the site of ventricular activation in a group of 12 patients undergoing electrophysiologic pacing studies. They acquired gated blood-pool studies during pacing at up to four sites at each of two different rates. A total of 50 studies were made. At a pacing rate of 100 beats/min, when the pacing electrode was the right-ventricular outflow tract, 7/8; at the anterolateral left-ventricular wall, 4/4. When the Fourier activation site was at the right-ventricular apex, 9/9 times the pacing electrode was there; at the right-ventricular outflow tract, 7/10; in the left ventricle, 4/4. Fourier analysis of gated blood-pool studies can help identify the site of ventricular activation but is not sufficiently accurate to fully replace endocardial mapping.

  19. Identification of two catalytic residues in RAG1 that define a single active site within the RAG1/RAG2 protein complex.

    PubMed

    Fugmann, S D; Villey, I J; Ptaszek, L M; Schatz, D G

    2000-01-01

    During V(D)J recombination, the RAG1 and RAG2 proteins cooperate to catalyze a series of DNA bond breakage and strand transfer reactions. The structure, location, and number of active sites involved in RAG-mediated catalysis have as yet not been determined. Using protein secondary structure prediction algorithms, we have identified a region of RAG1 with possible structural similarities to the active site regions of transposases and retroviral integrases. Based on this information, we have identified two aspartic acid residues in RAG1 (D600 and D708) that function specifically in catalysis. The results support a model in which RAG1 contains a single, divalent metal ion binding active site structurally related to the active sites of transposases/integrases and responsible for all catalytic functions of the RAG protein complex. PMID:10678172

  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. Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form*

    PubMed Central

    Gould, Christine M.; Antal, Corina E.; Reyes, Gloria; Kunkel, Maya T.; Adams, Ryan A.; Ziyar, Ahdad; Riveros, Tania; Newton, Alexandra C.

    2011-01-01

    Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C. PMID:21715334

  2. Educating for Political Activity: A Younger Generational Response

    ERIC Educational Resources Information Center

    Mac an Ghaill, Mairtin

    2010-01-01

    This paper is a response to Professor Chitty's "Educational Review" Guest Lecture article, "Educating for political activity". I address the three sections of his paper: a global and national-based politics of war, corporate manipulation and parliamentary scandals. This provides a basis to draw upon empirical material from a recent critical…

  3. Dynamics of lung macrophage activation in response to helminth infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most of our understanding of the development and phenotype of alternatively activated macrophages (AAM) has been obtained from studies investigating the response of bone marrow- and peritoneal-derived cells to IL-4 or IL-13 stimulation. Comparatively little is known about the development of the AAM...

  4. Sensorimotor-Independent Prefrontal Activity During Response Inhibition

    PubMed Central

    Cai, Weidong; Cannistraci, Christopher J.; Gore, John C.; Leung, Hoi-Chung

    2015-01-01

    A network of brain regions involving the ventral inferior frontal gyrus/anterior insula (vIFG/AI), presupplementary motor area (pre-SMA) and basal ganglia has been implicated in stopping impulsive, unwanted responses. However, whether this network plays an equal role in response inhibition under different sensorimotor contexts has not been tested systematically. Here, we conducted an fMRI experiment using the stop signal task, a sensorimotor task requiring occasional withholding of the planned response upon the presentation of a stop signal. We manipulated both the sensory modality of the stop signal (visual versus auditory) and the motor response modality (hand versus eye). Results showed that the vIFG/AI and the preSMA along with the right middle frontal gyrus were commonly activated in response inhibition across the various sensorimotor conditions. Our findings provide direct evidence for a common role of these frontal areas, but not striatal areas in response inhibition independent of the sensorimotor contexts. Nevertheless, these three frontal regions exhibited different activation patterns during successful and unsuccessful stopping. Together with the existing evidence, we suggest that the vIFG/AI is involved in the early stages of stopping such as triggering the stop process while the preSMA may play a role in regulating other cortical and subcortical regions involved in stopping. PMID:23798325

  5. Sensorimotor-independent prefrontal activity during response inhibition.

    PubMed

    Cai, Weidong; Cannistraci, Christopher J; Gore, John C; Leung, Hoi-Chung

    2014-05-01

    A network of brain regions involving the ventral inferior frontal gyrus/anterior insula (vIFG/AI), presupplementary motor area (pre-SMA) and basal ganglia has been implicated in stopping impulsive, unwanted responses. However, whether this network plays an equal role in response inhibition under different sensorimotor contexts has not been tested systematically. Here, we conducted an fMRI experiment using the stop signal task, a sensorimotor task requiring occasional withholding of the planned response upon the presentation of a stop signal. We manipulated both the sensory modality of the stop signal (visual versus auditory) and the motor response modality (hand versus eye). Results showed that the vIFG/AI and the preSMA along with the right middle frontal gyrus were commonly activated in response inhibition across the various sensorimotor conditions. Our findings provide direct evidence for a common role of these frontal areas, but not striatal areas in response inhibition independent of the sensorimotor contexts. Nevertheless, these three frontal regions exhibited different activation patterns during successful and unsuccessful stopping. Together with the existing evidence, we suggest that the vIFG/AI is involved in the early stages of stopping such as triggering the stop process while the preSMA may play a role in regulating other cortical and subcortical regions involved in stopping. PMID:23798325

  6. Phase response curves in the characterization of epileptiform activity

    NASA Astrophysics Data System (ADS)

    Perez Velazquez, J. L.; Galán, R. F.; Dominguez, L. Garcia; Leshchenko, Y.; Lo, S.; Belkas, J.; Erra, R. Guevara

    2007-12-01

    Coordinated cellular activity is a major characteristic of nervous system function. Coupled oscillator theory offers unique avenues to address cellular coordination phenomena. In this study, we focus on the characterization of the dynamics of epileptiform activity, based on some seizures that manifest themselves with very periodic rhythmic activity, termed absence seizures. Our approach consists in obtaining experimentally the phase response curves (PRCs) in the neocortex and thalamus, and incorporating these PRCs into a model of coupled oscillators. Phase preferences of the stationary states and their stability are determined, and these results from the model are compared with the experimental recordings, and interpreted in physiological terms.

  7. Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response

    NASA Astrophysics Data System (ADS)

    De Rosa, Lucia; Finetti, Federica; Diana, Donatella; di Stasi, Rossella; Auriemma, Sara; Romanelli, Alessandra; Fattorusso, Roberto; Ziche, Marina; Morbidelli, Lucia; D’Andrea, Luca Domenico

    2016-08-01

    The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor.

  8. Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response

    PubMed Central

    De Rosa, Lucia; Finetti, Federica; Diana, Donatella; Di Stasi, Rossella; Auriemma, Sara; Romanelli, Alessandra; Fattorusso, Roberto; Ziche, Marina; Morbidelli, Lucia; D’Andrea, Luca Domenico

    2016-01-01

    The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor. PMID:27498819

  9. Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response.

    PubMed

    De Rosa, Lucia; Finetti, Federica; Diana, Donatella; Di Stasi, Rossella; Auriemma, Sara; Romanelli, Alessandra; Fattorusso, Roberto; Ziche, Marina; Morbidelli, Lucia; D'Andrea, Luca Domenico

    2016-01-01

    The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor. PMID:27498819

  10. Bacterial lifestyle shapes the regulation of stringent response activation

    PubMed Central

    Boutte, Cara C.; Crosson, Sean

    2014-01-01

    Bacteria inhabit enormously diverse niches and have a correspondingly large array of regulatory mechanisms to adapt to often inhospitable and variable environments. The stringent response allows bacteria to quickly reprogram transcription in response to changes in nutrient availability. Although the proteins controlling this response are conserved in almost all bacterial species, recent work has illuminated considerable diversity in the starvation cues and regulatory mechanisms that activate stringent signaling proteins in bacteria from different environments. In this review we describe the signals and genetic circuitries that control the stringent signaling systems of a copiotroph, a bacteriovore, an oligotroph and a mammalian pathogen – Escherichia coli, Myxococcus xanthus, Caulobacter crescentus and Mycobacterium tuberculosis, respectively – and discuss how control of the stringent response in these species is adapted to their particular lifestyles. PMID:23419217

  11. Geographical matching of volatile signals and pollinator olfactory responses in a cycad brood-site mutualism.

    PubMed

    Suinyuy, Terence N; Donaldson, John S; Johnson, Steven D

    2015-10-01

    Brood-site mutualisms represent extreme levels of reciprocal specialization between plants and insect pollinators, raising questions about whether these mutualisms are mediated by volatile signals and whether these signals and insect responses to them covary geographically in a manner expected from coevolution. Cycads are an ancient plant lineage in which almost all extant species are pollinated through brood-site mutualisms with insects. We investigated whether volatile emissions and insect olfactory responses are matched across the distribution range of the African cycad Encephalartos villosus. This cycad species is pollinated by the same beetle species across its distribution, but cone volatile emissions are dominated by alkenes in northern populations, and by monoterpenes and a pyrazine compound in southern populations. In reciprocal choice experiments, insects chose the scent of cones from the local region over that of cones from the other region. Antennae of beetles from northern populations responded mainly to alkenes, while those of beetles from southern populations responded mainly to pyrazine. In bioassay experiments, beetles were most strongly attracted to alkenes in northern populations and to the pyrazine compound in southern populations. Geographical matching of cone volatiles and pollinator olfactory preference is consistent with coevolution in this specialized mutualism. PMID:26446814

  12. Guide to ground water remediation at CERCLA response action and RCRA corrective action sites

    SciTech Connect

    1995-10-01

    This Guide contains the regulatory and policy requirements governing remediation of ground water contaminated with hazardous waste [including radioactive mixed waste (RMW)], hazardous substances, or pollutants/contaminants that present (or may present) an imminent and substantial danger. It was prepared by the Office of Environmental Policy and Assistance, RCRA/CERCLA Division (EH-413), to assist Environmental Program Managers (ERPMs) who often encounter contaminated ground water during the performance of either response actions under CERCLA or corrective actions under Subtitle C of RCRA. The Guide begins with coverage of the regulatory and technical issues that are encountered by ERPM`s after a CERCLA Preliminary Assessment/Site Investigation (PA/SI) or the RCRA Facility Assessment (RFA) have been completed and releases into the environment have been confirmed. It is based on the assumption that ground water contamination is present at the site, operable unit, solid waste management unit, or facility. The Guide`s scope concludes with completion of the final RAs/corrective measures and a determination by the appropriate regulatory agencies that no further response action is necessary.

  13. Stochastic Seismic Response of an Algiers Site with Random Depth to Bedrock

    SciTech Connect

    Badaoui, M.; Mebarki, A.; Berrah, M. K.

    2010-05-21

    Among the important effects of the Boumerdes earthquake (Algeria, May 21{sup st} 2003) was that, within the same zone, the destructions in certain parts were more important than in others. This phenomenon is due to site effects which alter the characteristics of seismic motions and cause concentration of damage during earthquakes. Local site effects such as thickness and mechanical properties of soil layers have important effects on the surface ground motions.This paper deals with the effect of the randomness aspect of the depth to bedrock (soil layers heights) which is assumed to be a random variable with lognormal distribution. This distribution is suitable for strictly non-negative random variables with large values of the coefficient of variation. In this case, Monte Carlo simulations are combined with the stiffness matrix method, used herein as a deterministic method, for evaluating the effect of the depth to bedrock uncertainty on the seismic response of a multilayered soil. This study considers a P and SV wave propagation pattern using input accelerations collected at Keddara station, located at 20 km from the epicenter, as it is located directly on the bedrock.A parametric study is conducted do derive the stochastic behavior of the peak ground acceleration and its response spectrum, the transfer function and the amplification factors. It is found that the soil height heterogeneity causes a widening of the frequency content and an increase in the fundamental frequency of the soil profile, indicating that the resonance phenomenon concerns a larger number of structures.

  14. Stochastic Seismic Response of an Algiers Site with Random Depth to Bedrock

    NASA Astrophysics Data System (ADS)

    Badaoui, M.; Berrah, M. K.; Mébarki, A.

    2010-05-01

    Among the important effects of the Boumerdes earthquake (Algeria, May 21st 2003) was that, within the same zone, the destructions in certain parts were more important than in others. This phenomenon is due to site effects which alter the characteristics of seismic motions and cause concentration of damage during earthquakes. Local site effects such as thickness and mechanical properties of soil layers have important effects on the surface ground motions. This paper deals with the effect of the randomness aspect of the depth to bedrock (soil layers heights) which is assumed to be a random variable with lognormal distribution. This distribution is suitable for strictly non-negative random variables with large values of the coefficient of variation. In this case, Monte Carlo simulations are combined with the stiffness matrix method, used herein as a deterministic method, for evaluating the effect of the depth to bedrock uncertainty on the seismic response of a multilayered soil. This study considers a P and SV wave propagation pattern using input accelerations collected at Keddara station, located at 20 km from the epicenter, as it is located directly on the bedrock. A parametric study is conducted do derive the stochastic behavior of the peak ground acceleration and its response spectrum, the transfer function and the amplification factors. It is found that the soil height heterogeneity causes a widening of the frequency content and an increase in the fundamental frequency of the soil profile, indicating that the resonance phenomenon concerns a larger number of structures.

  15. Site-specific earthquake response analysis for Paducah Gaseous Diffusion Plant, Paducah, Kentucky. Final report

    SciTech Connect

    Sykora, D.W.; Davis, J.J.

    1993-08-01

    The Paducah Gaseous Diffusion Plant (PGDP), owned by the US Department of Energy (DOE) and operated under contract by Martin Marietta Energy systems, Inc., is located southwest of Paducah, Kentucky. An aerial photograph and an oblique sketch of the plant are shown in Figures 1 and 2, respectively. The fenced portion of the plant consists of 748 acres. This plant was constructed in the 1950`s and is one of only two gaseous diffusion plants in operation in the United States; the other is located near Portsmouth, Ohio. The facilities at PGDP are currently being evaluated for safety in response to natural seismic hazards. Design and evaluation guidelines to evaluate the effects of earthquakes and other natural hazards on DOE facilities follow probabilistic hazard models that have been outlined by Kennedy et al. (1990). Criteria also established by Kennedy et al. (1990) classify diffusion plants as ``moderate hazard`` facilities. The US Army Engineer Waterways Experiment Station (WES) was tasked to calculate the site response using site-specific design earthquake records developed by others and the results of previous geotechnical investigations. In all, six earthquake records at three hazard levels and four individual and one average soil columns were used.

  16. Anticipating Human Activities Using Object Affordances for Reactive Robotic Response.

    PubMed

    Koppula, Hema S; Saxena, Ashutosh

    2016-01-01

    An important aspect of human perception is anticipation, which we use extensively in our day-to-day activities when interacting with other humans as well as with our surroundings. Anticipating which activities will a human do next (and how) can enable an assistive robot to plan ahead for reactive responses. Furthermore, anticipation can even improve the detection accuracy of past activities. The challenge, however, is two-fold: We need to capture the rich context for modeling the activities and object affordances, and we need to anticipate the distribution over a large space of future human activities. In this work, we represent each possible future using an anticipatory temporal conditional random field (ATCRF) that models the rich spatial-temporal relations through object affordances. We then consider each ATCRF as a particle and represent the distribution over the potential futures using a set of particles. In extensive evaluation on CAD-120 human activity RGB-D dataset, we first show that anticipation improves the state-of-the-art detection results. We then show that for new subjects (not seen in the training set), we obtain an activity anticipation accuracy (defined as whether one of top three predictions actually happened) of 84.1, 74.4 and 62.2 percent for an anticipation time of 1, 3 and 10 seconds respectively. Finally, we also show a robot using our algorithm for performing a few reactive responses. PMID:26656575

  17. Philip Morris's health information web site appears responsible but undermines public health.

    PubMed

    Smith, Elizabeth A; Malone, Ruth E

    2008-01-01

    Many people may search for information about tobacco use, the largest cause of preventable mortality in the United States, on the Internet. In 1999, Philip Morris U.S.A. (PM), the country's biggest cigarette manufacturer, posted a Web site and launched a campaign to encourage people to obtain information about tobacco and health issues there. The company asserted that its goal was to deliver the messages of the public health community about tobacco. However, internal tobacco company documents reveal that the site was a public relations effort intended to help the company avoid punishment and regulation. Examination of the language on the Web site reveals many contradictions and omissions that may undermine public health messages. Among these are vague and confusing information about addiction, tar, and nicotine, a lack of motivators to quit smoking, and silence about tobacco-related mortality. By appearing to join with public health organizations in disseminating "responsible" messages about tobacco, PM may improve its image, thus facilitating its ability to continue to sell its lethal products. Public health nurses should be prepared to examine health information on the Internet for subtle biases, to counter PM's specific language about smoking to patients, and to challenge PM's larger corporate goals. PMID:18950420

  18. Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents.

    PubMed

    Hudson, Elton P; Eppler, Ross K; Beaudoin, Julianne M; Dordick, Jonathan S; Reimer, Jeffrey A; Clark, Douglas S

    2009-04-01

    The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, a(w). The apparent turnover number k(cat)(app) in these systems ranged from 0.2 to 80 s(-1) and increased 11-fold in isooctane and up to 50-fold in tetrahydrofuran with increasing a(w). (19)F NMR relaxation experiments employing an active-site inhibitor were used to assess the dependence of active-site motions on a(w). The rates of NMR-derived fast (k > 10(7) s(-1)) and slow (k < 10(4) s(-1)) active-site motions increased in both solvents upon hydration, but only the slow motions correlated with k(cat). The (19)F chemical shift was a sensitive probe of the local electronic environment and provided an empirical measure of the active-site dielectric constant epsilon(as), which increased with hydration to epsilon(as) approximately 13 in each solvent. In both solvents, the transition state free energy data and epsilon(as) followed Kirkwood's model for the continuum solvation of a dipole, indicating that water also enhanced catalysis by altering the active-site's electronic environment and increasing its polarity to better stabilize the transition state. These results reveal that favorable dynamic and electrostatic effects both contribute to accelerated catalysis by solubilized subtilisin Carlsberg upon hydration in organic solvents. PMID:19317505

  19. 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

  20. [Bone marrow stromal damage mediated by immune response activity].

    PubMed

    Vojinović, J; Kamenov, B; Najman, S; Branković, Lj; Dimitrijević, H

    1994-01-01

    The aim of this work was to estimate influence of activated immune response on hematopoiesis in vitro, using the experimental model of BCG immunized BALB/c mice and in patients with chronic immunoactivation: long-lasting infections, autoimmunity or malignancy. We correlated changes in long term bone marrow cultures (Dexter) and NBT reduction with appearance of anemia in patients and experimental model of immunization by BCG. Increased spontaneous NBT reduction pointed out role of macrophage activation in bone marrow stroma damage. Long-term bone marrow cultures showed reduced number of hematopoietic cells, with predomination of fibroblasts and loss of fat cells. This results correlated with anemia and leucocytosis with stimulated myelopoiesis in peripheral blood. Activation of immune response, or acting of any agent that directly changes extracellular matrix and cellularity of bone marrow, may result in microenviroment bone marrow damage that modify hematopoiesis. PMID:18173180

  1. Humpback Dolphin (Genus Sousa) Behavioural Responses to Human Activities.

    PubMed

    Piwetz, Sarah; Lundquist, David; Würsig, Bernd

    2015-01-01

    Humpback dolphins (genus Sousa) use shallow, near-shore waters throughout their range. This coastal distribution makes them vulnerable to recreational and commercial disturbances, especially near heavily populated and industrialized areas. Most research focusing on Sousa and human activities has emphasized direct impacts and threats, involving injury and death, with relatively little focus on indirect effects on dolphins, such as changes in behaviour that may lead to deleterious effects. Understanding behaviour is important in resolving human-wildlife conflict and is an important component of conservation. This chapter gives an overview of animal behavioural responses to human activity with examples from diverse taxa; reviews the scientific literature on behavioural responses of humpback dolphins to human activity throughout their range, including marine vessel traffic, dolphin tourism, cetacean-fishery interactions, noise pollution, and habitat alteration; and highlights information and data gaps for future humpback dolphin research to better inform behaviour-based management decisions that contribute to conservation efforts. PMID:26555621

  2. The value of SPECT/CT in localizing pain site and prediction of treatment response in patients with chronic low back pain.

    PubMed

    Lee, Inki; Budiawan, Hendra; Moon, Jee Youn; Cheon, Gi Jeong; Kim, Yong Chul; Paeng, Jin Chul; Kang, Keon Wook; Chung, June-Key; Lee, Dong Soo

    2014-12-01

    In many circumstances, causing sites of low back pain (LBP) cannot be determined only by anatomical imaging. Combined functional and morphological imaging such as bone scan with single-photon emission computed tomography/computed tomography (SPECT/CT) may be helpful in identifying active lesions. The purpose of this study was to evaluate the usefulness of bone SPECT/CT in localizing the pain site and the treatment of chronic LBP. One hundred seventy-five patients suffering from chronic LBP who underwent SPECT/CT were included, retrospectively. All of the patients received multiple general treatments according to the symptoms, and some of them underwent additional target-specific treatment based on SPECT/CT. Numerical rating scale (NRS) pain score was used to assess the pain intensity. Of 175 patients, 127 showed good response to the given therapies, while the rest did not. Overall, 79.4% of patients with definite active lesions showed good response. Patients with mild active or no lesions on SPECT/CT had relatively lower response rate of 63.0%. Good response was observed by the treatment with the guidance of active lesions identified on SPECT/CT. SPECT/CT could be useful in identifying active lesions in patients with chronic LBP and guiding the clinicians to use adequate treatment. PMID:25469075

  3. 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.

  4. A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.

    PubMed

    Méthot, Nathalie; Vaillancourt, John P; Huang, JingQi; Colucci, John; Han, Yongxin; Ménard, Stéphane; Zamboni, Robert; Toulmond, Sylvie; Nicholson, Donald W; Roy, Sophie

    2004-07-01

    Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies. PMID:15067000

  5. CD83 Modulates B Cell Activation and Germinal Center Responses.

    PubMed

    Krzyzak, Lena; Seitz, Christine; Urbat, Anne; Hutzler, Stefan; Ostalecki, Christian; Gläsner, Joachim; Hiergeist, Andreas; Gessner, André; Winkler, Thomas H; Steinkasserer, Alexander; Nitschke, Lars

    2016-05-01

    CD83 is a maturation marker for dendritic cells. In the B cell lineage, CD83 is expressed especially on activated B cells and on light zone B cells during the germinal center (GC) reaction. The function of CD83 during GC responses is unclear. CD83(-/-) mice have a strong reduction of CD4(+) T cells, which makes it difficult to analyze a functional role of CD83 on B cells during GC responses. Therefore, in the present study we generated a B cell-specific CD83 conditional knockout (CD83 B-cKO) model. CD83 B-cKO B cells show defective upregulation of MHC class II and CD86 expression and impaired proliferation after different stimuli. Analyses of GC responses after immunization with various Ags revealed a characteristic shift in dark zone and light zone B cell numbers, with an increase of B cells in the dark zone of CD83 B-cKO mice. This effect was not accompanied by alterations in the level of IgG immune responses or by major differences in affinity maturation. However, an enhanced IgE response was observed in CD83 B-cKO mice. Additionally, we observed a strong competitive disadvantage of CD83-cKO B cells in GC responses in mixed bone marrow chimeras. Furthermore, infection of mice with Borrelia burgdorferi revealed a defect in bacterial clearance of CD83 B-cKO mice with a shift toward a Th2 response, indicated by a strong increase in IgE titers. Taken together, our results show that CD83 is important for B cell activation and modulates GC composition and IgE Ab responses in vivo. PMID:26983787

  6. 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

  7. 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

  8. Delphinid behavioral responses to incidental mid-frequency active sonar.

    PubMed

    Henderson, E Elizabeth; Smith, Michael H; Gassmann, Martin; Wiggins, Sean M; Douglas, Annie B; Hildebrand, John A

    2014-10-01

    Opportunistic observations of behavioral responses by delphinids to incidental mid-frequency active (MFA) sonar were recorded in the Southern California Bight from 2004 through 2008 using visual focal follows, static hydrophones, and autonomous recorders. Sound pressure levels were calculated between 2 and 8 kHz. Surface behavioral responses were observed in 26 groups from at least three species of 46 groups out of five species encountered during MFA sonar incidents. Responses included changes in behavioral state or direction of travel, changes in vocalization rates and call intensity, or a lack of vocalizations while MFA sonar occurred. However, 46% of focal groups not exposed to sonar also changed their behavior, and 43% of focal groups exposed to sonar did not change their behavior. Mean peak sound pressure levels when a behavioral response occurred were around 122 dB re: 1 μPa. Acoustic localizations of dolphin groups exhibiting a response gave insight into nighttime movement patterns and provided evidence that impacts of sonar may be mediated by behavioral state. The lack of response in some cases may indicate a tolerance of or habituation to MFA sonar by local populations; however, the responses that occur at lower received levels may point to some sensitization as well. PMID:25324099

  9. 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

  10. Characterizing wind turbine system response to lightning activity

    SciTech Connect

    McNiff, B.; LaWhite, N.; Muljadi, E.

    1998-07-01

    A lightning protection research program was instituted by National Renewable Energy Laboratory to minimize lightning damage to wind turbines and to further the understanding of effective damage mitigation techniques. To that end, a test program is under way to observe lightning activity, protection system response, and damage at a wind power plant in the Department of Energy (DOE) and Electric Power Research Institute (EPRI) Turbine Verification Program. The authors installed Lightning activated surveillance cameras along with a special storm tracking device to observe the activity in the wind plant area. They instrumented the turbines with lightning and ground current detection devices to log direct and indirect strike activity at each unit. They installed a surge monitor on the utility interface to track incoming activity from the transmission lines. Maintenance logs are used to verify damage and determine downtime and repair costs. Actual strikes to turbines were recorded on video and ancillary devices. The test setup and some results are discussed in this paper.

  11. 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

  12. Active-site mobility revealed by the crystal structure of arylmalonate decarboxylase from Bordetella bronchiseptica.

    PubMed

    Kuettner, E Bartholomeus; Keim, Antje; Kircher, Markus; Rosmus, Susann; Sträter, Norbert

    2008-03-21

    Arylmalonate decarboxylase (AMDase) from Bordetella bronchiseptica catalyzes the enantioselective decarboxylation of arylmethylmalonates without the need for an organic cofactor or metal ion. The decarboxylation reaction is of interest for the synthesis of fine chemicals. As basis for an analysis of the catalytic mechanism of AMDase and for a rational enzyme design, we determined the X-ray structure of the enzyme up to 1.9 A resolution. Like the distantly related aspartate or glutamate racemases, AMDase has an aspartate transcarbamoylase fold consisting of two alpha/beta domains related by a pseudo dyad. However, the domain orientation of AMDase differs by about 30 degrees from that of the glutamate racemases, and also significant differences in active-site structures are observed. In the crystals, four independent subunits showing different conformations of active-site loops are present. This finding is likely to reflect the active-site mobility necessary for catalytic activity. PMID:18258259

  13. 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.

  14. Comment and response document for the long-term surveillance plan for the Collins Ranch Disposal Site Lakeview, Oregon

    SciTech Connect

    Not Available

    1993-11-01

    Twenty-nine comments from the US Nuclear Regulatory Commission and six from the Grand Junction Project Office for the long-term surveillance plan for the Collins Ranch Disposal Site, Lakeview, Oregon are documented along with their corresponding responses.

  15. Comment and response document for the long-term surveillance plan for the Shiprock, New Mexico, disposal site

    SciTech Connect

    Not Available

    1994-09-01

    This document contains comments and responses regarding the long-term surveillance plan for the Shiprock, New Mexico uranium mine tailings disposal site. Discrepencies and errors within the plan document are noted and corrections are recorded.

  16. Comment and response document for the long-term surveillance plan for the Bodo Canyon Disposal Site, Durango. Colorado

    SciTech Connect

    1996-07-01

    This document contains comments made by the US Nuclear Regulatory Commission upon their review of the Long-Term Surveillance Plan for the Bodo Canyon Disposal Site, Durango, Colorado. Responses to the comments are also included in the document.

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2015-01-01

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis. DOI: http://dx.doi.org/10.7554/eLife.11620.001 PMID:26673079

  19. Leptin in nucleus of the solitary tract alters the cardiovascular responses to aortic baroreceptor activation.

    PubMed

    Ciriello, John

    2013-06-01

    Recent data suggests that neurons expressing the long form of the leptin receptor form at least two distinct groups within the caudal nucleus of the solitary tract (NTS): a group within the lateral NTS (Slt) and one within the medial (Sm) and gelantinosa (Sg) NTS. Discrete injections of leptin into Sm and Sg, a region that receives chemoreceptor input, elicit increases in arterial pressure (AP) and renal sympathetic nerve activity (RSNA). However, the effect of microinjections of leptin into Slt, a region that receives baroreceptor input is unknown. Experiments were done in the urethane-chloralose anesthetized, paralyzed and artificially ventilated Wistar or Zucker obese rat to determine leptin's effect in Slt on heart rate (HR), AP and RSNA during electrical stimulation of the aortic depressor nerve (ADN). Depressor sites within Slt were first identified by the microinjection of l-glutamate (Glu; 0.25M; 10nl) followed by leptin microinjections. In the Wistar rat leptin microinjection (50ng; 20nl) into depressor sites within the lateral Slt elicited increases in HR and RSNA, but no changes in AP. Additionally, leptin injections into Slt prior to Glu injections at the same site or to stimulation of the ADN were found to attenuate the decreases in HR, AP and RSNA to both the Glu injection and ADN stimulation. In Zucker obese rats, leptin injections into NTS depressor sites did not elicit cardiovascular responses, nor altered the cardiovascular responses elicited by stimulation of ADN. Those data suggest that leptin acts at the level of NTS to alter the activity of neurons that mediate the cardiovascular responses to activation of the aortic baroreceptor reflex. PMID:23535030

  20. Site response assessment at the city of Alkhobar, Eastern Saudi Arabia from microtremor and boring data

    NASA Astrophysics Data System (ADS)

    Fnais, M. S.

    2014-12-01

    The AlKhobar City affected by distant earthquakes from Zagros fold-fault belt of subduction zone. These earthquakes produced great site effects on the sedimentary layers that in turn significantly influenced earthquake ground motions in the area. Mapping of site response using microtremor measurements compared with geological and borehole data of Alkhobar city is the main target of this work. The resonance frequency and their H/V values have been calculated using Nakamura technique through deployed of seismograph instruments at 113 sites in AlKhobar city for different time periods. The recording length was about one hour with sampling frequency of 100 Hz. Most of the measured sites present three peaks for the resonance frequency; the first peak ranges from 0.33 to 1.03 Hz, the second peak ranges from 1.03 to 1.23 Hz, while the third peak ranges from 1.23-1.73. Tests have been conducted to ensure that these peaks are natural in origin. The northern zones of AlKhobar City have lower values of resonance frequency indicating great thickness of sediments. In contrast, the southern parts of the city have higher values of resonance frequency illustrating shallow depths of the bedrock. Furthermore, twenty of boreholes have been conducted through AlKhobar City to different depth. Standard penetration test (SPT) data has been corrected and used to calculate the resonance frequency at their locations. Borehole results clarified that the resonance frequency values range from 0.27 to 1.95 Hz. These results are correlated well with that of the microtremor measurements. Accordingly, the first peak have been interpreted due to the impedance contrast between the limestone and the overlying sediments, while the third peak is originated from a boundary between the upper most surface layer and the underlying sediments. These results must be applied for earthquake risk mitigation in AlKhobar City.

  1. Identification and Validation of Novel Hedgehog-Responsive Enhancers Predicted by Computational Analysis of Ci/Gli Binding Site Density

    PubMed Central

    Richards, Neil; Parker, David S.; Johnson, Lisa A.; Allen, Benjamin L.; Barolo, Scott; Gumucio, Deborah L.

    2015-01-01

    The Hedgehog (Hh) signaling pathway directs a multitude of cellular responses during embryogenesis and adult tissue homeostasis. Stimulation of the pathway results in activation of Hh target genes by the transcription factor Ci/Gli, which binds to specific motifs in genomic enhancers. In Drosophila, only a few enhancers (patched, decapentaplegic, wingless, stripe, knot, hairy, orthodenticle) have been shown by in vivo functional assays to depend on direct Ci/Gli regulation. All but one (orthodenticle) contain more than one Ci/Gli site, prompting us to directly test whether homotypic clustering of Ci/Gli binding sites is sufficient to define a Hh-regulated enhancer. We therefore developed a computational algorithm to identify Ci/Gli clusters that are enriched over random expectation, within a given region of the genome. Candidate genomic regions containing Ci/Gli clusters were functionally tested in chicken neural tube electroporation assays and in transgenic flies. Of the 22 Ci/Gli clusters tested, seven novel enhancers (and the previously known patched enhancer) were identified as Hh-responsive and Ci/Gli-dependent in one or both of these assays, including: Cuticular protein 100A (Cpr100A); invected (inv), which encodes an engrailed-related transcription factor expressed at the anterior/posterior wing disc boundary; roadkill (rdx), the fly homolog of vertebrate Spop; the segment polarity gene gooseberry (gsb); and two previously untested regions of the Hh receptor-encoding patched (ptc) gene. We conclude that homotypic Ci/Gli clustering is not sufficient information to ensure Hh-responsiveness; however, it can provide a clue for enhancer recognition within putative Hedgehog target gene loci. PMID:26710299

  2. Insight into the mechanism of phosphoenolpyruvate mutase catalysis derived from site-directed mutagenesis studies of active site residues.

    PubMed

    Jia, Y; Lu, Z; Huang, K; Herzberg, O; Dunaway-Mariano, D

    1999-10-26

    PEP mutase catalyzes the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate in biosynthetic pathways leading to phosphonate secondary metabolites. A recent X-ray structure [Huang, K., Li, Z., Jia, Y., Dunaway-Mariano, D., and Herzberg, O. (1999) Structure (in press)] of the Mytilus edulis enzyme complexed with the Mg(II) cofactor and oxalate inhibitor reveals an alpha/beta-barrel backbone-fold housing an active site in which Mg(II) is bound by the two carboxylate groups of the oxalate ligand and the side chain of D85 and, via bridging water molecules, by the side chains of D58, D85, D87, and E114. The oxalate ligand, in turn, interacts with the side chains of R159, W44, and S46 and the backbone amide NHs of G47 and L48. Modeling studies identified two feasible PEP binding modes: model A in which PEP replaces oxalate with its carboxylate group interacting with R159 and its phosphoryl group positioned close to D58 and Mg(II) shifting slightly from its original position in the crystal structure, and model B in which PEP replaces oxalate with its phosphoryl group interacting with R159 and Mg(II) retaining its original position. Site-directed mutagenesis studies of the key mutase active site residues (R159, D58, D85, D87, and E114) were carried out in order to evaluate the catalytic roles predicted by the two models. The observed retention of low catalytic activity in the mutants R159A, D85A, D87A, and E114A, coupled with the absence of detectable catalytic activity in D58A, was interpreted as evidence for model A in which D58 functions in nucleophilic catalysis (phosphoryl transfer), R159 functions in PEP carboxylate group binding, and the carboxylates of D85, D87 and E114 function in Mg(II) binding. These results also provide evidence against model B in which R159 serves to mediate the phosphoryl transfer. A catalytic motif, which could serve both the phosphoryl transfer and the C-C cleavage enzymes of the PEP mutase superfamily, is proposed. PMID:10571990

  3. Active seismic and passive microtremor HVSR for assessing site effects in Jammu city, NW Himalaya, India—A case study

    NASA Astrophysics Data System (ADS)

    Mahajan, A. K.; Mundepi, A. K.; Chauhan, Neetu; Jasrotia, A. S.; Rai, Nitesh; Gachhayat, Tapas Kumar

    2012-02-01

    1-D shear wave velocity structure is important for site effect studies and geotechnical engineering, but it is quite difficult and expensive to derive from the conventional geophysical techniques. Active (MASW) and passive (microtremors, HVSR) methods were conducted at 30 sites in the frontal part of the Himalaya which is characterized by soft sediments and strong seismological effects. Shear wave velocity ( Vs) in the range of ~ 238 m/s to ~ 450 m/s has been obtained from 30 m thick layer of quaternary sediments overlying Lower Miocene bed rock (Upper Siwalik Conglomerate) in Jammu city, NW Himalaya. The shear wave velocity ( Vs) along with seismic input motion of Chamoli earthquake (mb 6.8) has been used to obtain site response spectrum. The response spectrum suggests five to seven times increase in peak ground acceleration for single or two storey buildings and by eight to twelve times increase in amplification ratio with respect to input ground motion. The amplification spectrum shows peak amplification of ~ 2 Hz-~ 3 Hz in the central part and ~ 1.75 Hz-2 Hz in the northern, southwestern and southeastern parts of the city. The advantage of microtremor HVSR is that it yields direct estimate of the fundamental frequency which is found to vary from ~ 1 Hz to ~ 3 Hz for same sites. Further, the 1-D velocity models obtained from ModelHVSR Matlab routine have been compared with the soil models prepared by derived using MASW. The comparison shows correlation between soil models for sites having high shallow impedance contrast between the overlying sediments and very stiff material (bedrock) underneath as than sites having less impedance contrast.

  4. Mutational analysis of acute-phase response factor/Stat3 activation and dimerization.

    PubMed Central

    Sasse, J; Hemmann, U; Schwartz, C; Schniertshauer, U; Heesel, B; Landgraf, C; Schneider-Mergener, J; Heinrich, P C; Horn, F

    1997-01-01

    Signal transducer and transcription (STAT) factors are activated by tyrosine phosphorylation in response to a variety of cytokines, growth factors, and hormones. Tyrosine phosphorylation triggers dimerization and nuclear translocation of these transcription factors. In this study, the functional role of carboxy-terminal portions of the STAT family member acute-phase response factor/Stat3 in activation, dimerization, and transactivating potential was analyzed. We demonstrate that truncation of 55 carboxy-terminal amino acids causes constitutive activation of Stat3 in COS-7 cells, as is known for the Stat3 isoform Stat3beta. By the use of deletion and point mutants, it is shown that both carboxy- and amino-terminal portions of Stat3 are involved in this phenomenon. Dimerization of Stat3 was blocked by point mutations affecting residues both in the vicinity of the tyrosine phosphorylation site (Y705) and more distant from this site, suggesting that multiple interactions are involved in dimer formation. Furthermore, by reporter gene assays we demonstrate that carboxy-terminally truncated Stat3 proteins are incapable of transactivating an interleukin-6-responsive promoter in COS-7 cells. In HepG2 hepatoma cells, however, these truncated Stat3 forms transmit signals from the interleukin-6 signal transducer gp130 equally well as does full-length Stat3. We conclude that, dependent on the cell type, different mechanisms allow Stat3 to regulate target gene transcription either with or without involvement of its putative carboxy-terminal transactivation domain. PMID:9234724

  5. The prolactin responses to active and passive heating in man.

    PubMed

    Low, David; Purvis, Alison; Reilly, Thomas; Cable, N Tim

    2005-11-01

    The aim of this study was to compare the prolactin and blood pressure responses at identical core temperatures during active and passive heat stresses, using prolactin as an indirect marker of central fatigue. Twelve male subjects cycled to exhaustion at 60% maximal oxygen uptake (VO2peak) in a room maintained at 33 degrees C (active). In a second trial they were passively heated (passive) in a water bath (41.56 +/- 1.65 degrees C) until core temperature was equal to the core temperature observed at exhaustion during the active trial. Blood samples were taken from an indwelling venous cannula for the determination of serum prolactin during active heating and at corresponding core temperatures during passive heating. Core temperature was not significantly different between the two methods of heating and averaged 38.81 +/- 0.53 and 38.82 +/- 0.70 degrees C (data expressed as means +/- s.d.) at exhaustion during active heating and at the end of passive heating, respectively (P > 0.05). Mean arterial blood pressure was significantly lower throughout passive heating (active, 73 +/- 9 mmHg; passive, 62 +/- 12 mmHg; P < 0.01). Despite the significantly reduced blood pressure responses during passive heating, during both forms of heating the prolactin response was the same (active, 14.9 +/- 12.6 ng ml(-1); passive, 13.3 +/- 9.6 ng ml(-1); n.s.). These results suggest that thermoregulatory, i.e. core temperature, and not cardiovascular afferents provide the key stimulus for the release of prolactin, an indirect marker of central fatigue, during exercise in the heat. PMID:16157657

  6. Predicting Nonlinear Site Response Using Spectral Acceleration Vs PGV/Vs30: A Case History Using the Volvi-Test Site

    NASA Astrophysics Data System (ADS)

    Guéguen, Philippe

    2015-12-01

    In this study, we analyze the efficiency of the ratio between particle velocity and shear wave velocity as a strain proxy for evaluating the nonlinear seismic response of sediments. The in situ stress-strain relationships are derived from accelerometric vertical array recordings at the TST site in Volvi (Thessaloniki, Greece). First, the shear wave velocity between two successive sensors was computed by seismic interferometry and strain was computed as the velocity ratio or the relative displacement between sensors. The shear-wave velocity profile and in situ shear modulus degradation curve with strain were compared with previous studies performed at the TST site. Finally, the stress-strain relationships were derived from data recorded at the surface by extending the strain proxy and stress values to the ratio between peak ground velocity and the Vs30 parameter used for site classification, i.e. without requiring the accelerometric vertical array. Our model captures the in situ nonlinear response of the site, without consideration of azimuth or distance of the earthquakes. In conclusion, the acceleration (stress) values, based on the accelerometric response spectra instead of peak ground acceleration compared with the deformation (strain) proxy, provide an effective model of the in situ nonlinear response, providing information that can be integrated into ground motion prediction equations.

  7. Predicting Nonlinear Site Response Using Spectral Acceleration Vs PGV/Vs30: A Case History Using the Volvi-Test Site

    NASA Astrophysics Data System (ADS)

    Guéguen, Philippe

    2016-06-01

    In this study, we analyze the efficiency of the ratio between particle velocity and shear wave velocity as a strain proxy for evaluating the nonlinear seismic response of sediments. The in situ stress-strain relationships are derived from accelerometric vertical array recordings at the TST site in Volvi (Thessaloniki, Greece). First, the shear wave velocity between two successive sensors was computed by seismic interferometry and strain was computed as the velocity ratio or the relative displacement between sensors. The shear-wave velocity profile and in situ shear modulus degradation curve with strain were compared with previous studies performed at the TST site. Finally, the stress-strain relationships were derived from data recorded at the surface by extending the strain proxy and stress values to the ratio between peak ground velocity and the Vs30 parameter used for site classification, i.e. without requiring the accelerometric vertical array. Our model captures the in situ nonlinear response of the site, without consideration of azimuth or distance of the earthquakes. In conclusion, the acceleration (stress) values, based on the accelerometric response spectra instead of peak ground acceleration compared with the deformation (strain) proxy, provide an effective model of the in situ nonlinear response, providing information that can be integrated into ground motion prediction equations.

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

    PubMed

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

    2012-05-01

    Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds. PMID:21877154

  9. Underground Test Area Activity Quality Assurance Plan Nevada National Security Site, Nevada. Revision 1

    SciTech Connect

    Farnham, Irene; Krenzien, Susan

    2012-10-01

    This Quality Assurance Plan (QAP) provides the overall quality assurance (QA) requirements and general quality practices to be applied to the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office (NNSA/NSO) Underground Test Area (UGTA) activities. The requirements in this QAP are consistent with DOE Order 414.1C, Quality Assurance (DOE, 2005); U.S. Environmental Protection Agency (EPA) Guidance for Quality Assurance Project Plans for Modeling (EPA, 2002); and EPA Guidance on the Development, Evaluation, and Application of Environmental Models (EPA, 2009). NNSA/NSO, or designee, must review this QAP every two years. Changes that do not affect the overall scope or requirements will not require an immediate QAP revision but will be incorporated into the next revision cycle after identification. Section 1.0 describes UGTA objectives, participant responsibilities, and administrative and management quality requirements (i.e., training, records, procurement). Section 1.0 also details data management and computer software requirements. Section 2.0 establishes the requirements to ensure newly collected data are valid, existing data uses are appropriate, and environmental-modeling methods are reliable. Section 3.0 provides feedback loops through assessments and reports to management. Section 4.0 provides the framework for corrective actions. Section 5.0 provides references for this document.

  10. Structure of the E. Coli Bifunctional GlmU Acetyltransferase Active Site with Substrates and Products

    SciTech Connect

    Olsen,L.; Vetting, M.; Roderick, S.

    2007-01-01

    The biosynthesis of UDP-GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA-dependent acetylation of GlcN-1-PO{sub 4} to form GlcNAc-1-PO{sub 4} and its subsequent condensation with UTP to form pyrophosphate and UDP-GlcNAc. As a metabolite, UDP-GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl-CoA, CoA/GlcN-1-PO{sub 4}, and desulpho-CoA/GlcNAc-1-PO{sub 4}. These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2-amino group of GlcN-1-PO{sub 4} is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism.

  11. Active sites of ligand-protected Au25 nanoparticle catalysts for CO2 electroreduction to CO

    NASA Astrophysics Data System (ADS)

    Alfonso, Dominic R.; Kauffman, Douglas; Matranga, Christopher

    2016-05-01

    Recent experimental studies have reported the electrochemical reduction of carbon dioxide (CO2) into CO at atomically precise negatively charged Au25- nanoclusters. The studies showed CO2 conversion at remarkably low overpotentials, but the exact mechanisms and nature of the active sites remain unclear. We used first-principles density functional theory and continuum solvation models to examine the role of the cluster during electrochemical CO2 reduction and analyze the free energies of proposed intermediate species. Contrary to previous assumptions, our results show that the fully ligand protected cluster is not an active CO2 reduction catalyst because formation of the crucial carboxyl intermediate required very high electrochemical potentials. Instead, our calculations suggest that the reduction process likely occurs on a dethiolated gold site, and adsorbed carboxyl intermediate formation was significantly stabilized at dethiolated gold sites. These findings point to the crucial role of exposed metal sites during electrochemical CO2 reduction at gold nanocluster catalysts.

  12. Active sites of ligand-protected Au25 nanoparticle catalysts for CO2 electroreduction to CO.

    PubMed

    Alfonso, Dominic R; Kauffman, Douglas; Matranga, Christopher

    2016-05-14

    Recent experimental studies have reported the electrochemical reduction of carbon dioxide (CO2) into CO at atomically precise negatively charged Au25 (-) nanoclusters. The studies showed CO2 conversion at remarkably low overpotentials, but the exact mechanisms and nature of the active sites remain unclear. We used first-principles density functional theory and continuum solvation models to examine the role of the cluster during electrochemical CO2 reduction and analyze the free energies of proposed intermediate species. Contrary to previous assumptions, our results show that the fully ligand protected cluster is not an active CO2 reduction catalyst because formation of the crucial carboxyl intermediate required very high electrochemical potentials. Instead, our calculations suggest that the reduction process likely occurs on a dethiolated gold site, and adsorbed carboxyl intermediate formation was significantly stabilized at dethiolated gold sites. These findings point to the crucial role of exposed metal sites during electrochemical CO2 reduction at gold nanocluster catalysts. PMID:27179498

  13. Differential responses to artificial selection on oviposition site preferences in Drosophila melanogaster and D. simulans.

    PubMed

    Soto, Eduardo M; Betti, María I L; Hurtado, Juan; Hasson, Esteban

    2015-12-01

    The preference-performance relationship in plant-insect interactions is a central theme in evolutionary ecology. Among many insects, eggs are vulnerable and larvae have limited mobility, making the choice of an appropriate oviposition site one of the most important decisions for a female. We investigated the evolution of oviposition preferences in Drosophila melanogaster Meigen and Drosophila simulans Sturtevant by artificially selecting for the preference for 2 natural resources, grape and quince. The main finding of our study is the differential responses of D. melanogaster and D. simulans. Although preferences evolved in the experimental populations of D. melanogaster, responses were not consistent with the selection regimes applied. In contrast, responses in D. simulans were consistent with expectations, demonstrating that this species has selectable genetic variation for the trait. Furthermore, crosses between D. simulans divergent lines showed that the genetic factors involved in grape preference appear to be largely recessive. In summary, our artificial selection study suggests that D. melanogaster and D. simulans possess different genetic architectures for this trait. PMID:25263841

  14. RpoS proteolysis is regulated by a mechanism that does not require the SprE (RssB) response regulator phosphorylation site.

    PubMed

    Peterson, Celeste N; Ruiz, Natividad; Silhavy, Thomas J

    2004-11-01

    In Escherichia coli the response regulator SprE (RssB) facilitates degradation of the sigma factor RpoS by delivering it to the ClpXP protease. This process is regulated: RpoS is degraded in logarithmic phase but becomes stable upon carbon starvation, resulting in its accumulation. Because SprE contains a CheY domain with a conserved phosphorylation site (D58), the prevailing model posits that this control is mediated by phosphorylation. To test this model, we mutated the conserved response regulator phosphorylation site (D58A) of the chromosomal allele of sprE and monitored RpoS levels in response to carbon starvation. Though phosphorylation contributed to the SprE basal activity, we found that RpoS proteolysis was still regulated upon carbon starvation. Furthermore, our results indicate that phosphorylation of wild-type SprE occurs by a mechanism that is independent of acetyl phosphate. PMID:15489452

  15. Long-term active layer and ground surface temperature trends: results of 12 years of observations at Alaskan CALM sites

    NASA Astrophysics Data System (ADS)

    Shiklomanov, N. I.; Nelson, F. E.; Streletskyi, D. A.; Klene, A. E.; Schimek, M.; Little, J.

    2006-12-01

    The uppermost layer of seasonal thawing above permafrost (the active layer) is an important regulator of energy and mass fluxes between the surface and the atmosphere in the polar regions. The Circumpolar Active Layer Monitoring (CALM) program is a network of sites at which data about active-layer thickness (ALT) and dynamics are collected. CALM was established in the 1990s to observe and detect the long-term response of the active layer and near-surface permafrost to changes in climate. Active layer monitoring is an important component of efforts to assess the effects of global change in permafrost environments. CALM strategies are evolving; this presentation showcases some additions to CALM observation procedures designed to monitor processes and detect changes not anticipated in the original CALM protocol of the early 1990s. In this study we used data from 12 (1995-2006) years of extensive, spatially oriented field observations at CALM sites in northern Alaska to examine landscape-specific spatial and temporal trends in active-layer thickness and air and ground surface temperature. Despite an observed increase in air temperature, active-layer thickness exhibited a decreasing trend over the study period. This result indicates that soil consolidation accompanying penetration of thaw into an ice-rich stratum at the base of the active layer has resulted in subsidence of the surface with little or no apparent thickening of the active layer, as traditionally defined. Differential Global Positioning Systems (DGPS) technology was used to detect frost heave and thaw settlement within representative landscapes. Preliminary results indicate that heave and settlement follow patterns of spatial variation similar to those of active-layer thickness. To evaluate the effect of vegetation on ground surface temperature, several heat-transfer coefficients were estimated, including land cover specific thermal diffusivity and empirical n-factors.

  16. Role of activation in alveolar macrophage-mediated suppression of the plaque-forming cell response.

    PubMed Central

    Mbawuike, I N; Herscowitz, H B

    1988-01-01

    Alveolar macrophages (AM) are highly suppressive of the in vitro plaque-forming cell (PFC) response of spleen cells obtained from mice primed with sheep erythrocytes. Comparison of macrophage populations obtained from disparate anatomical sites revealed that although in both cases there was a cell-concentration-dependent suppression of the PFC response, resident AM or AM activated as a result of intravenous injection of Mycobacterium bovis BCG were equally suppressive at the doses examined. Although there was a similar dose-dependent suppression with peritoneal macrophages, BCG-activated cells were more suppressive of the PFC response than were resident cells. In contrast, splenic macrophages at comparable concentrations were not at all suppressive. Resident AM exhibited significantly lower levels of 5'-nucleotidase activity than did resident peritoneal macrophages. Macrophage-mediated suppression of the in vitro PFC response could not be attributed to the release of toxic oxygen metabolites (H2O2, O2- ,and .OH) or prostaglandins, since the addition of catalase, superoxide dismutase, 2-mercaptoethanol, or indomethacin did not completely reverse suppression. These results suggest that the lung microenvironment may maintain AM in an activated state which contributes to their potential immunoregulatory functions. PMID:2830191

  17. Brx Mediates the Response of Lymphocytes to Osmotic Stress Through the Activation of NFAT5

    PubMed Central

    Kino, Tomoshige; Takatori, Hiroaki; Manoli, Irini; Wang, Yonghong; Tiulpakov, Anatoly; Blackman, Marc R.; Su, Yan A.; Chrousos, George P.; DeCherney, Alan H.; Segars, James H.

    2010-01-01

    Extracellular hyperosmolarity, or osmotic stress, generally caused by differences in salt and macromolecule concentrations across the plasma membrane, occurs in lymphoid organs and at inflammatory sites. The response of immune cells to osmotic stress is regulated by nuclear factor of activated T cells 5 (NFAT5), a transcription factor that induces the expression of hyperosmolarity-responsive genes and stimulates cytokine production. We report that the guanine nucleotide exchange factor (GEF) Brx [also known as protein kinase A–anchoring protein 13 (AKAP13)] is essential for the expression of nfat5 in response to osmotic stress, thus transmitting the extracellular hyperosmolarity signal and enabling differentiation of splenic B cells and production of immunoglobulin. This process required the activity of p38 mitogen-activated protein kinase (MAPK) and NFAT5 and involved a physical interaction between Brx and c-Jun N-terminal kinase (JNK)–interacting protein 4 (JIP4), a scaffold molecule specific to activation of the p38 MAPK cascade. Our results indicate that Brx integrates the responses of immune cells to osmotic stress and inflammation by elevating intracellular osmolarity and stimulating the production of cytokines. PMID:19211510

  18. Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase.

    PubMed Central

    Milgrom, Y M; Murataliev, M B; Boyer, P D

    1998-01-01

    Experiments are reported on the uni-site catalysis and the transition from uni-site to multi-site catalysis with bovine heart mitochondrial F1-ATPase. The very slow uni-site ATP hydrolysis is shown to occur without tightly bound nucleotides present and with or without Pi in the buffer. Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates. They provide rate constant information, and show that an apparent Km for a second site of about 2 microM and Vmax of 10 s-1, as suggested by others, is not operative. Careful initial velocity measurements also eliminate other suggested Km values and are consistent with bi-site activation to near maximal hydrolysis rates, with a Km of about 130 microM and Vmax of about 700 s-1. However, the results do not eliminate the possibility of additional 'hidden' Km values with similar Vmax:Km ratios. Recent data on competition between TNP-ATP and ATP revealed a third catalytic site for ATP in the millimolar concentration range. This result, and those reported in the present paper, allow the conclusion that the mitochondrial F1-ATPase can attain near maximal activity in bi-site catalysis. Our data also add to the evidence that a recent claim, that the mitochondrial F1-ATPase does not show catalytic site cooperativity, is invalid. PMID:9480927

  19. Response of winter birds to soil remediation along the Columbia River at the Hanford Site

    SciTech Connect

    Becker, James M.; McKinstry, Craig A.

    2004-04-01

    The Columbia River at the Hanford Site, located in south-central Washington State, USA, is a regionally important refugium for overwintering birds. Some of the river shoreline has been designated by the U.S. Department of Energy for environmental clean-up following past production of materials for nuclear weapons. We evaluated the effects of soil remediation on winter birds at six inactive nuclear reactor areas. Remediation activities consisted of daily excavation and removal of approximately 1,035 t of contaminated soil from previously herbicided and denuded areas located between 30 m and 400 m and mostly in line-of-sight of the river shoreline. Remediation activities had no apparent effect on numbers of riverine or terrestrial birds using adjacent undisturbed shoreline and riparian habitat.

  20. Response of winter birds to soil remediation along the Columbia River at the Hanford Site.

    PubMed

    Becker, J M; McKinstry, C A

    2004-01-01

    The Columbia River at the Hanford Site, located in south-central Washington State, U.S.A., is a regionally important refugium for overwintering birds. Some of the river shoreline has been designated by the U.S. Department of Energy for environmental clean-up following past production of materials for nuclear weapons. We evaluated the effects of soil remediation on winter birds at six inactive nuclear reactor areas. Remediation activities consisted of daily excavation and removal of approximately 1035 t of contaminated soil from previously herbicided and denuded areas located between 30 and 400 m and mostly in line-of-sight of the river shoreline. Remediation activities had no apparent effect on numbers of riverine or terrestrial birds using adjacent undisturbed shoreline and riparian habitat. PMID:15074620

  1. 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.

  2. Modified Active Site Coordination in a Clinical Mutant of Sulfite Oxidase

    SciTech Connect

    Doonan, C.J.; Wilson, H.L.; Rajagopalan, K.V.; Garrett, R.M.; Bennett, B.; Prince, R.C.; George, G.N.

    2009-06-02

    The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme sulfite oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.

  3. Changes in thyroid peroxidase activity in response to various chemicals.

    PubMed

    Song, Mee; Kim, Youn-Jung; Park, Yong-Keun; Ryu, Jae-Chun

    2012-08-01

    Thyroperoxidase (TPO) is a large heme-containing glycoprotein that catalyzes the transfer of iodine to thyroglobulin during thyroid hormone (TH) synthesis. Previously, we established an in vitro assay for TPO activity based on human recombinant TPO (hrTPO) stably transfected into human follicular thyroid carcinoma (FTC-238) cells. It is important to determine whether environmental chemicals can disrupt TPO activity because it is an important factor in the TH axis. In this study, we used our assay to examine the changes in TPO activity in response to various chemicals, including benzophenones (BPs), polycyclic aromatic hydrocarbons (PAHs), and persistent organic pollutants (POPs). Overall, BPs, PAHs, and POPs slightly altered TPO activity at low doses, as compared with the positive controls methimazole (MMI), genistein, and 2,2',4,4'-tetrahydroxy BP. Benzophenone, benzhydrol, 3-methylchloranthracene, pyrene, benzo(k)fluoranthene, benzo(e)pyrene, perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), and heptachlor decreased TPO activity, while 2,4-dihydroxy BP, 2,2'-dihydroxy-4-methoxy BP, and dibenzo(a,h)anthracene increased TPO activity. From these data, we can predict the disruption of TPO activity by various chemicals as a sensitive TH end point. TPO activity should be considered when enacting measures to regulate environmental exposure to thyroid-disrupting chemicals. PMID:22699773

  4. β-Glucuronidase activity and mitochondrial dysfunction: the sites where flavonoid glucuronides act as anti-inflammatory agents

    PubMed Central

    Kawai, Yoshichika

    2014-01-01

    Epidemiological and experimental studies suggest that the consumption of flavonoid-rich diets decreases the risk of various chronic diseases such as cardiovascular diseases. Although studies on the bioavailability of flavonoids have been well-characterized, the tissue and cellular localizations underlying their biological mechanisms are largely unknown. The development and application of novel monoclonal antibodies revealed that macrophages could be the major target of dietary flavonoids in vivo. Using macrophage-like cell lines in vitro, we examined the molecular basis of the interaction between the macrophages and flavonoids, especially the glucuronide metabolites. We have found that extracellular β-glucuronidase secreted from macrophages is essential for the bioactivation of the glucuronide conjugates into the aglycone, and that the enzymatic activity, which requires an acidic pH, is promoted by the increased secretion of lactate in response to the mitochondrial dysfunction. This review describes our recent findings indicating the molecular mechanisms responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites. We propose that the extracellular activity of β-glucuronidase associated with the status of the mitochondrial function in the target cells might be important biomarkers for the specific sites where the glucuronides of dietary flavonoids can act as anti-atherosclerotic and anti-inflammatory agents in vivo. PMID:24895476

  5. 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

  6. Coordinate regulation/localization of the carbohydrate responsive binding protein (ChREBP) by two nuclear export signal sites: Discovery of a new leucine-rich nuclear export signal site

    SciTech Connect

    Fukasawa, Masashi; Ge, Qing; Wynn, R. Max; Ishii, Seiji; Uyeda, Kosaku

    2010-01-08

    Carbohydrate response element binding protein (ChREBP) is responsible for conversion of dietary carbohydrate to storage fat in liver by coordinating expression of the enzymes that channel glycolytic pyruvate into lipogenesis. The activation of ChREBP in response to high glucose is nuclear localization and transcription, and the inactivation of ChREBP under low glucose involves export from the nucleus to the cytosol. Here we report a new nuclear export signal site ('NES1') of ChREBP. Together these signals provide ChREBP with two NES sequences, both the previously reported NES2 and now the new NES1 coordinate to interact together with CRM1 (exportin) for nuclear export of the carbohydrate response element binding protein.

  7. Response Activation in Overlapping Tasks and the Response-Selection Bottleneck

    ERIC Educational Resources Information Center

    Schubert, Torsten; Fischer, Rico; Stelzel, Christine

    2008-01-01

    The authors investigated the impact of response activation on dual-task performance by presenting a subliminal prime before the stimulus in Task 2 (S2) of a psychological refractory period (PRP) task. Congruence between prime and S2 modulated the reaction times in Task 2 at short stimulus onset asynchrony despite a PRP effect. This Task 2…

  8. Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents

    PubMed Central

    Hudson, Elton P; Eppler, Ross K; Beaudoin, Julianne M; Dordick, Jonathan S; Reimer, Jeffrey A; Clark, Douglas S

    2009-01-01

    The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, aw. The apparent turnover number kcatapp in these systems ranged from 0.2 to 80 s−1 and increased 11-fold in isooctane and up to 50-fold in tetrahydrofuran with increasing aw. 19F-NMR relaxation experiments employing an active-site inhibitor were used to assess the dependence of active-site motions on aw. The rates of NMR-derived fast (k > 107 s−1) and slow (k < 104 s−1) active-site motions increased in both solvents upon hydration, but only the slow motions correlated with kcat. The 19F chemical shift was a sensitive probe of the local electronic environment and provided an empirical measure of the active-site dielectric constant εas, which increased with hydration to εas ≈ 13 in each solvent. In both solvents the transition state free energy data and εas followed Kirkwood’s model for the continuum solvation of a dipole, indicating that water also enhanced catalysis by altering the active-site’s electronic environment and increasing its polarity to better stabilize the transition state. These results reveal that favorable dynamic and electrostatic effects both contribute to accelerated catalysis by solubilized subtilisin Carlsberg upon hydration in organic solvents. PMID:19317505

  9. Acylpeptide hydrolase: inhibitors and some active site residues of the human enzyme.

    PubMed

    Scaloni, A; Jones, W M; Barra, D; Pospischil, M; Sassa, S; Popowicz, A; Manning, L R; Schneewind, O; Manning, J M

    1992-02-25

    Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions. PMID:1740429

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

    PubMed

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

    2015-03-01

    Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed. PMID:25613522

  11. Reduced brain activation in violent adolescents during response inhibition

    PubMed Central

    Qiao, Yi; Mei, Yi; Du, XiaoXia; Xie, Bin; Shao, Yang

    2016-01-01

    Deficits in inhibitory control have been linked to aggression and violent behaviour. This study aimed to observe whether violent adolescents show different brain activation patterns during response inhibition and to ascertain the roles these brain regions play. A self-report method and modified overt aggression scale (MOAS) were used to evaluate violent behaviour. Functional magnetic resonance imaging was performed in 22 violent adolescents and 17 matched healthy subjects aged 12 to 18 years. While scanning, a go/no-go task was performed. Between-group comparisons revealed that activation in the bilateral middle and superior temporal gyrus, hippocampus, and right orbitofrontal area (BA11) regions were significantly reduced in the violent group compared with the control group. Meanwhile, the violent group had more widespread activation in the prefrontal cortex than that observed in the control group. Activation of the prefrontal cortex in the violent group was widespread but lacking in focus, failing to produce intensive activation in some functionally related regions during response inhibition. PMID:26888566

  12. 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.

  13. Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site

    PubMed Central

    Wang, Lu; Fried, Stephen D.; Boxer, Steven G.; Markland, Thomas E.

    2014-01-01

    Enzymes use protein architectures to create highly specialized structural motifs that can greatly enhance the rates of complex chemical transformations. Here, we use experiments, combined with ab initio simulations that exactly include nuclear quantum effects, to show that a triad of strongly hydrogen-bonded tyrosine residues within the active site of the enzyme ketosteroid isomerase (KSI) facilitates quantum proton delocalization. This delocalization dramatically stabilizes the deprotonation of an active-site tyrosine residue, resulting in a very large isotope effect on its acidity. When an intermediate analog is docked, it is incorporated into the hydrogen-bond network, giving rise to extended quantum proton delocalization in the active site. These results shed light on the role of nuclear quantum effects in the hydrogen-bond network that stabilizes the reactive intermediate of KSI, and the behavior of protons in biological systems containing strong hydrogen bonds. PMID:25503367

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

    PubMed Central

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

    2014-01-01

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

  15. Summary of canister overheating incident at the Carbon Tetrachloride Expedited Response Action site

    SciTech Connect

    Driggers, S.A.

    1994-03-10

    The granular activated carbon (GAC)-filled canister that overheated was being used to adsorb carbon tetrachloride vapors drawn from a well near the 216-Z-9 Trench, a subsurface disposal site in the 200 West Area of the Hanford Site. The overheating incident resulted in a band of discolored paint on the exterior surface of the canister. Although there was no other known damage to equipment, no injuries to operating personnel, and no releases of hazardous materials, the incident is of concern because it was not anticipated. It also poses the possibility of release of carbon tetrachloride and other hazardous vapors if the incident were to recur. All soil vapor extraction system (VES) operations were halted until a better understanding of the cause of the incident could be determined and controls implemented to reduce the possibility of a recurrence. The focus of this report and the intent of all the activities associated with understanding the overheating incident has been to provide information that will allow safe restart of the VES operations, develop operational limits and controls to prevent recurrence of an overheating incident, and safely optimize recovery of carbon tetrachloride from the ground.

  16. Effects of three activities on annoyance responses to recorded flyovers

    NASA Technical Reports Server (NTRS)

    Gunn, W. J.; Shepherd, W. T.; Fletcher, J. L.

    1975-01-01

    Subjects participated in an experiment in which they were engaged in TV viewing, telephone listening, or reverie (no activity) for a 1/2-hour session. During the session, they were exposed to a series of recorded aircraft sounds at the rate of one flight every 2 minutes. Within each session, four levels of flyover noise, separated by 5dB increments, were presented several times in a Latin Square balanced sequence. The peak level of the noisiest flyover in any session was fixed at 95, 90, 85, 75, or 70 dBA. At the end of the test session, subjects recorded their responses to the aircraft sounds, using a bipolar scale which covered the range from 'very pleasant' to 'extremely annoying'. Responses to aircraft noises were found to be significantly affected by the particular activity in which the subjects were engaged. Furthermore, not all subjects found the aircraft sounds to be annoying.

  17. Activation and Regulation of DNA-Driven Immune Responses

    PubMed Central

    2015-01-01

    SUMMARY The innate immune system provides early defense against infections and also plays a key role in monitoring alterations of homeostasis in the body. DNA is highly immunostimulatory, and recent advances in this field have led to the identification of the innate immune sensors responsible for the recognition of DNA as well as the downstream pathways that are activated. Moreover, information on how cells regulate DNA-driven immune responses to avoid excessive inflammation is now emerging. Finally, several reports have demonstrated how defects in DNA sensing, signaling, and regulation are associated with susceptibility to infections or inflammatory diseases in humans and model organisms. In this review, the current literature on DNA-stimulated innate immune activation is discussed, and important new questions facing this field are proposed. PMID:25926682

  18. Hemodynamic responses to functional activation accessed by optical imaging

    NASA Astrophysics Data System (ADS)

    Ni, Songlin; Li, Pengcheng; Yang, Yuanyuan; Lv, Xiaohua; Luo, Qingming

    2006-01-01

    A multi-wavelength light-emitting diode (LED) and laser diode (LD) based optical imaging system was developed to visualize the changes in cerebral blood flow, oxygenation following functional activation simultaneously in rodent cortex. The 2-D blood flow image was accessed by laser speckle contrast imaging, and the spectroscopic imaging of intrinsic signal was used for the calculation of oxyhemoglobin (HbO), deoxyhemoglobin (Hb) and total hemoglobin (HbT) concentration. The combination of spectroscopic imaging and laser speckle contrast imaging provides the capability to simultaneously investigate the spatial and temporal blood flow and hemoglobin concentration changes with high resolution, which may lead to a better understanding of the coupling between neuronal activation and vascular responses. The optical imaging system been built is compact and convenient to investigators. And it is reliable to acquire raw data. In present study, the hemodynamic responses to cortical spreading depression (CSD) in parietal cortex of ~-chloralose/urethan anesthetized rats were demonstrated.

  19. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    PubMed

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst. PMID:27402448

  20. 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

  1. Photodynamic therapy for cancer and activation of immune response

    NASA Astrophysics Data System (ADS)

    Mroz, Pawel; Huang, Ying-Ying; Hamblin, Michael R.

    2010-02-01

    Anti-tumor immunity is stimulated after PDT for cancer due to the acute inflammatory response, exposure and presentation of tumor-specific antigens, and induction of heat-shock proteins and other danger signals. Nevertheless effective, powerful tumor-specific immune response in both animal models and also in patients treated with PDT for cancer, is the exception rather than the rule. Research in our laboratory and also in others is geared towards identifying reasons for this sub-optimal immune response and discovering ways of maximizing it. Reasons why the immune response after PDT is less than optimal include the fact that tumor-antigens are considered to be self-like and poorly immunogenic, the tumor-mediated induction of CD4+CD25+foxP3+ regulatory T-cells (T-regs), that are able to inhibit both the priming and the effector phases of the cytotoxic CD8 T-cell anti-tumor response and the defects in dendritic cell maturation, activation and antigen-presentation that may also occur. Alternatively-activated macrophages (M2) have also been implicated. Strategies to overcome these immune escape mechanisms employed by different tumors include combination regimens using PDT and immunostimulating treatments such as products obtained from pathogenic microorganisms against which mammals have evolved recognition systems such as PAMPs and toll-like receptors (TLR). This paper will cover the use of CpG oligonucleotides (a TLR9 agonist found in bacterial DNA) to reverse dendritic cell dysfunction and methods to remove the immune suppressor effects of T-regs that are under active study.

  2. 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.

  3. Activity-dependent labeling of oxygenase enzymes in a trichloroethene-contaminated groundwater site.

    PubMed

    Lee, M Hope; Clingenpeel, Scott C; Leiser, Owen P; Wymore, Ryan A; Sorenson, Kent S; Watwood, Mary E

    2008-05-01

    A variety of naturally occurring bacteria produce enzymes that cometabolically degrade trichloroethene (TCE), including organisms with aerobic oxygenases. Groundwater contaminated with TCE was collected from the aerobic region of the Test Area North site of the Idaho National Laboratory. Samples were evaluated with enzyme activity probes, and resulted in measurable detection of toluene oxygenase activity (6-79% of the total microbial cells). Wells from both inside and outside contaminated plume showed activity. Toluene oxygenase-specific PCR primers determined that toluene-degrading genes were present in all groundwater samples evaluated. In addition, bacterial isolates were obtained and possessed toluene oxygenase enzymes, demonstrated activity, and were dominated by the phylotype Pseudomonas. This study demonstrated, through the use of enzymatic probes and oxygenase gene identification, that indigenous microorganisms at a contaminated site were cometabolically active. Documentation such as this can be used to substantiate observations of natural attenuation of TCE-contaminated groundwater plumes. PMID:17904715

  4. DNA damage processing by human 8-oxoguanine-DNA glycosylase mutants with the occluded active site.

    PubMed

    Lukina, Maria V; Popov, Alexander V; Koval, Vladimir V; Vorobjev, Yuri N; Fedorova, Olga S; Zharkov, Dmitry O

    2013-10-01

    8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation. PMID:23955443

  5. 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.

  6. 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

  7. Autophagic activity dictates the cellular response to oncogenic RAS

    PubMed Central

    Wang, Yihua; Wang, Xiao Dan; Lapi, Eleonora; Sullivan, Alexandra; Jia, Wei; He, You-Wen; Ratnayaka, Indrika; Zhong, Shan; Goldin, Robert D.; Goemans, Christoph G.; Tolkovsky, Aviva M.; Lu, Xin

    2012-01-01

    RAS is frequently mutated in human cancers and has opposing effects on autophagy and tumorigenesis. Identifying determinants of the cellular responses to RAS is therefore vital in cancer research. Here, we show that autophagic activity dictates the cellular response to oncogenic RAS. N-terminal Apoptosis-stimulating of p53 protein 2 (ASPP2) mediates RAS-induced senescence and inhibits autophagy. Oncogenic RAS-expressing ASPP2(Δ3/Δ3) mouse embryonic fibroblasts that escape senescence express a high level of ATG5/ATG12. Consistent with the notion that autophagy levels control the cellular response to oncogenic RAS, overexpressing ATG5, but not autophagy-deficient ATG5 mutant K130R, bypasses RAS-induced senescence, whereas ATG5 or ATG3 deficiency predisposes to it. Mechanistically, ASPP2 inhibits RAS-induced autophagy by competing with ATG16 to bind ATG5/ATG12 and preventing ATG16/ATG5/ATG12 formation. Hence, ASPP2 modulates oncogenic RAS-induced autophagic activity to dictate the cellular response to RAS: to proliferate or senesce. PMID:22847423

  8. Dynamical theory of active cellular response to external stress.

    PubMed

    De, Rumi; Safran, Samuel A

    2008-09-01

    We present a comprehensive, theoretical treatment of the orientational response to external stress of active, contractile cells embedded in a gel-like elastic medium. The theory includes both the forces that arise from the deformation of the matrix as well as forces due to the internal regulation of the stress fibers and focal adhesions of the cell. We calculate the time-dependent response of both the magnitude and the direction of the elastic dipole that characterizes the active forces exerted by the cell, for various situations. For static or quasistatic external stress, cells orient parallel to the stress while for high frequency dynamic external stress, cells orient nearly perpendicular. Both numerical and analytical calculations of these effects are presented. In addition we predict the relaxation time for the cellular response for both slowly and rapidly varying external stresses; several characteristic scaling regimes for the relaxation time as a function of applied frequency are predicted. We also treat the case of cells for which the regulation of the stress fibers and focal adhesions is controlled by strain (instead of stress) and show that the predicted dependence of the cellular orientation on the Poisson ratio of the matrix can differentiate strain vs stress regulation of cellular response. PMID:18851081

  9. Dynamical theory of active cellular response to external stress

    NASA Astrophysics Data System (ADS)

    de, Rumi; Safran, Samuel A.

    2008-09-01

    We present a comprehensive, theoretical treatment of the orientational response to external stress of active, contractile cells embedded in a gel-like elastic medium. The theory includes both the forces that arise from the deformation of the matrix as well as forces due to the internal regulation of the stress fibers and focal adhesions of the cell. We calculate the time-dependent response of both the magnitude and the direction of the elastic dipole that characterizes the active forces exerted by the cell, for various situations. For static or quasistatic external stress, cells orient parallel to the stress while for high frequency dynamic external stress, cells orient nearly perpendicular. Both numerical and analytical calculations of these effects are presented. In addition we predict the relaxation time for the cellular response for both slowly and rapidly varying external stresses; several characteristic scaling regimes for the relaxation time as a function of applied frequency are predicted. We also treat the case of cells for which the regulation of the stress fibers and focal adhesions is controlled by strain (instead of stress) and show that the predicted dependence of the cellular orientation on the Poisson ratio of the matrix can differentiate strain vs stress regulation of cellular response.

  10. Kinetics, dose response, tachyphylaxis and cross-tachyphylaxis of vascular leakage induced by endotoxin, zymosan-activated plasma and platelet-activating factor in the horse.

    PubMed

    Mills, P C; Ng, J C; Seawright, A A; Auer, D E

    1995-06-01

    Vascular leakage induced by intradermal injection of endotoxin, zymosan-activated plasma (ZAP) and platelet-activating factor (PAF) was measured in nine Thoroughbreds using 125-iodine human serum albumin (125I-HSA) as a marker in the blood. ZAP and PAF produced dose-dependent increases in vascular permeability with the maximum occurring within the first 15 min after injection. The vascular leakage induced by endotoxin was also dose-dependent, but the maximum occurred 2 h after intradermal injection. Intradermal sites previously injected with endotoxin were refractory to a second injection of endotoxin for up to 5 days. However, sites injected with endotoxin and re-injected with either ZAP or PAF remained responsive with increased vascular leakage compared to saline injected control sites re-injected with either ZAP or PAF. Diminished response to endotoxin challenge may contribute to the poor prognosis of endotoxaemia in the horse. PMID:7674456

  11. Modeling learning in brain stem and cerebellar sites responsible for VOR plasticity

    NASA Technical Reports Server (NTRS)

    Quinn, K. J.; Didier, A. J.; Baker, J. F.; Peterson, B. W.

    1998-01-01

    A simple model of vestibuloocular reflex (VOR) function was used to analyze several hypotheses currently held concerning the characteristics of VOR plasticity. The network included a direct vestibular pathway and an indirect path via the cerebellum. An optimization analysis of this model suggests that regulation of brain stem sites is critical for the proper modification of VOR gain. A more physiologically plausible learning rule was also applied to this network. Analysis of these simulation results suggests that the preferred error correction signal controlling gain modification of the VOR is the direct output of the accessory optic system (AOS) to the vestibular nuclei vs. a signal relayed through the cerebellum via floccular Purkinje cells. The potential anatomical and physiological basis for this conclusion is discussed, in relation to our current understanding of the latency of the adapted VOR response.

  12. SOFTWARE QUALITY ASSURANCE FOR EMERGENCY RESPONSE CONSEQUENCE ASSESSMENT MODELS AT DOE'S SAVANNAH RIVER SITE

    SciTech Connect

    Hunter, C

    2007-12-17

    The Savannah River National Laboratory's (SRNL) Atmospheric Technologies Group develops, maintains, and operates computer-based software applications for use in emergency response consequence assessment at DOE's Savannah River Site. These applications range from straightforward, stand-alone Gaussian dispersion models run with simple meteorological input to complex computational software systems with supporting scripts that simulate highly dynamic atmospheric processes. A software quality assurance program has been developed to ensure appropriate lifecycle management of these software applications. This program was designed to meet fully the overall structure and intent of SRNL's institutional software QA programs, yet remain sufficiently practical to achieve the necessary level of control in a cost-effective manner. A general overview of this program is described.

  13. Modeling learning in brain stem and cerebellar sites responsible for VOR plasticity.

    PubMed

    Quinn, K J; Didier, A J; Baker, J F; Peterson, B W

    1998-07-01

    A simple model of vestibuloocular reflex (VOR) function was used to analyze several hypotheses currently held concerning the characteristics of VOR plasticity. The network included a direct vestibular pathway and an indirect path via the cerebellum. An optimization analysis of this model suggests that regulation of brain stem sites is critical for the proper modification of VOR gain. A more physiologically plausible learning rule was also applied to this network. Analysis of these simulation results suggests that the preferred error correction signal controlling gain modification of the VOR is the direct output of the accessory optic system (AOS) to the vestibular nuclei vs. a signal relayed through the cerebellum via floccular Purkinje cells. The potential anatomical and physiological basis for this conclusion is discussed, in relation to our current understanding of the latency of the adapted VOR response. PMID:9671263

  14. Activation of the DNA Damage Response by RNA Viruses.

    PubMed

    Ryan, Ellis L; Hollingworth, Robert; Grand, Roger J

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  15. Activation of the DNA Damage Response by RNA Viruses

    PubMed Central

    Ryan, Ellis L.; Hollingworth, Robert; Grand, Roger J.

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  16. Monitoring of geological activity on astronomical sites of the Canary Islands, Hawaii, and Chile

    NASA Astrophysics Data System (ADS)

    Eff-Darwich, Antonio; Garcia-Lorenzo, Begoña; Rodriguez-Losada, Jose A.; Hernández-Gutiérrez, Luis E.; de la Nuez, Julio; Romero-Ruiz, Maria C.

    2009-09-01

    Future large and extremely large ground-based telescopes will demand stable geological settings.Remote sensing could be an unvaluable tool to analyse the impact of geological activity at selected astronomical sites, namely the observatories of El Teide (Tenerife, Canary Islands), Roque de los Muchachos (La Palma, Canary Islands), Mauna Kea (Hawaii) and Paranal (Chile; the candidate site of Cerro Ventarrones, Chile). In this sense, the extent of lava flows, eruptive clouds or ground deformation associated to seismic and/or volcanic activity could be analysed and characterised through remote sensing.

  17. 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

  18. 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

  19. 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.

  20. Small activating RNA binds to the genomic target site in a seed-region-dependent manner

    PubMed Central

    Meng, Xing; Jiang, Qian; Chang, Nannan; Wang, Xiaoxia; Liu, Chujun; Xiong, Jingwei; Cao, Huiqing; Liang, Zicai

    2016-01-01

    RNA activation (RNAa) is the upregulation of gene expression by small activating RNAs (saRNAs). In order to investigate the mechanism by which saRNAs act in RNAa, we used the progesterone receptor (PR) gene as a model, established a panel of effective saRNAs and assessed the involvement of the sense and antisense strands of saRNA in RNAa. All active saRNAs had their antisense strand effectively incorporated into Ago2, whereas such consistency did not occur for the sense strand. Using a distal hotspot for saRNA targeting at 1.6-kb upstream from the PR transcription start site, we further established that gene activation mediated by saRNA depended on the complementarity of the 5′ region of the antisense strand, and that such activity was largely abolished by mutations in this region of the saRNA. We found markedly reduced RNAa effects when we created mutations in the genomic target site of saRNA PR-1611, thus providing evidence that RNAa depends on the integrity of the DNA target. We further demonstrated that this saRNA bound the target site on promoter DNA. These results demonstrated that saRNAs work via an on-site mechanism by binding to target genomic DNA in a seed-region-dependent manner, reminiscent of miRNA-like target recognition. PMID:26873922

  1. A Ty1 Reverse Transcriptase Active-Site Aspartate Mutation Blocks Transposition but Not Polymerization†

    PubMed Central

    Uzun, Ozcan; Gabriel, Abram

    2001-01-01

    Reverse transcriptases (RTs) are found in a wide variety of mobile genetic elements including viruses, retrotransposons, and infectious organellar introns. An invariant triad of aspartates is thought to be required for the catalytic function of RTs. We generated RT mutants in the yeast retrotransposon Ty1, changing each of these active-site aspartates to asparagine or glutamate. All but one of the mutants lacked detectable polymerase activity. The novel exception, D211N, retained near wild-type in vitro polymerase activity within virus-like particles but failed to carry out in vivo transposition. For this mutant, minus-strand synthesis is impaired and formation of the plus-strand strong-stop intermediate is eliminated. Intragenic second-site suppressor mutations of the transposition defect map to the RNase H domain of the enzyme. Our results demonstrate that one of the three active-site aspartates in a retrotransposon RT is not catalytically critical. This implies a basic difference in the polymerase active-site geometry of Ty1 and human immunodeficiency virus RT and shows that subtle mutations in one domain can cause dramatic functional effects on a distant domain of the same enzyme. PMID:11413300

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

    PubMed

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

    2009-04-01

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

  3. Transcriptional regulation of the PXR gene: identification and characterization of a functional peroxisome proliferator-activated receptor alpha binding site within the proximal promoter of PXR.

    PubMed

    Aouabdi, Sihem; Gibson, Gordon; Plant, Nick

    2006-01-01

    The pregnane X receptor (PXR, NR1I2) is widely regarded as a central factor in the body's response to changes in the fluxome, the overall metabolite profile in the body. PXR expression is regulated by a number of chemicals at the transcriptional level; the majority of these chemicals are ligands for PXR and substrates for PXR target genes. However, transcriptional activators of PXR, such as clofibrate, do not seem to be PXR ligands or substrates for its target genes. Understanding the molecular mechanisms underlying both these expected and, more importantly, unexpected transcriptional activations is central to fully understanding the roles of PXR in the human body. We have carried out an in silico analysis of the human PXR proximal promoter, identifying putative protein/DNA interaction sites within the 2 kilobases (kb) 5' to the putative transcription start site. These sites included several for liver-enriched transcription factors, such as the hepatic nuclear factors and CAAT-enhancer binding protein alpha, and chicken ovalbumin upstream promoter transcription factor, commensurate with the high expression of PXR in liver. Furthermore, we identified putative binding sites for a number of ligand-activated transcription factors, suggesting that these factors may regulate PXR gene expression. Further analysis of this regulatory region has shown that transcriptional activation of PXR by peroxisome proliferator-activated receptor alpha (PPARalpha) is via a binding site located approximately 1.3 kb upstream of the putative transcription start site, with ablation of this site preventing PPARalpha-mediated activation of PXR gene expression. We present a model of how regulation of PXR gene expression by ligand-activated transcription factors may play a central role in the body's response to xenobiotic exposure. PMID:16243957

  4. Placebo-Activated Neural Systems are Linked to Antidepressant Responses

    PubMed Central

    Peciña, Marta; Bohnert, Amy S. B.; Sikora, Magdalena; Avery, Erich T.; Langenecker, Scott A.; Mickey, Brian J.; Zubieta, Jon-Kar

    2016-01-01

    Importance High placebo responses have been observed across a wide range of pathologies, severely impacting drug development. Objective Here we examined neurochemical mechanisms underlying the formation of placebo effects in patients with Major Depressive Disorder (MDD). Participants Thirty-five medication-free MDD patients. Design and Intervention We performed a single-blinded two-week cross-over randomized controlled trial of two identical oral placebos (described as having either “active” or “inactive” fast-acting antidepressant-like effects) followed by a 10-week open-label treatment with a selective serotonin reuptake inhibitor (SSRI) or in some cases, another agent as clinically indicated. The volunteers were studied with PET and the μ-opioid receptor (MOR)-selective radiotracer [11C]carfentanil after each 1-week “inactive” and “active” oral placebo treatment. In addition, 1 mL of isotonic saline was administered intravenously (i.v.) within sight of the volunteer during PET scanning every 4 min over 20 min only after the 1-week active placebo treatment, with instructions that the compound may be associated with the activation of brain systems involved in mood improvement. This challenge stimulus was utilized to test the individual capacity to acutely activate endogenous opioid neurotransmision under expectations of antidepressant effect. Setting A University Health System. Main Outcomes and Measures Changes in depressive symptoms in response to “active” placebo and antidepressant. Baseline and activation measures of MOR binding. Results Higher baseline MOR binding in the nucleus accumbens (NAc) was associated with better response to antidepressant treatment (r=0.48; p=0.02). Reductions in depressive symptoms after 1-week of “active” placebo treatment, compared to the “inactive”, were associated with increased placebo-induced μ-opioid neurotransmission in a network of regions implicated in emotion, stress regulation, and the

  5. A facile reflux procedure to increase active surface sites form highly active and durable supported p