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Sample records for active site position

  1. Nucleosome positioning and kinetics near transcription-start-site barriers are controlled by interplay between active remodeling and DNA sequence.

    PubMed

    Parmar, Jyotsana J; Marko, John F; Padinhateeri, Ranjith

    2014-01-01

    We investigate how DNA sequence, ATP-dependent chromatin remodeling and nucleosome-depleted 'barriers' co-operate to determine the kinetics of nucleosome organization, in a stochastic model of nucleosome positioning and dynamics. We find that 'statistical' positioning of nucleosomes against 'barriers', hypothesized to control chromatin structure near transcription start sites, requires active remodeling and therefore cannot be described using equilibrium statistical mechanics. We show that, unlike steady-state occupancy, DNA site exposure kinetics near a barrier is dominated by DNA sequence rather than by proximity to the barrier itself. The timescale for formation of positioning patterns near barriers is proportional to the timescale for active nucleosome eviction. We also show that there are strong gene-to-gene variations in nucleosome positioning near barriers, which are eliminated by averaging over many genes. Our results suggest that measurement of nucleosome kinetics can reveal information about sequence-dependent regulation that is not apparent in steady-state nucleosome occupancy.

  2. Site-specific mutagenesis and functional analysis of active sites of sulfur oxygenase reductase from Gram-positive moderate thermophile Sulfobacillus acidophilus TPY.

    PubMed

    Zhang, Huijun; Guo, Wenbin; Xu, Changan; Zhou, Hongbo; Chen, Xinhua

    2013-12-14

    Sequence alignments revealed that the conserved motifs of SORSa which formed an independent branch between archaea and Gram-negative bacteria SORs according to the phylogenetic relationship were similar with the archaea and Gram-negative bacteria SORs. In order to investigate the active sites of SORSa, cysteines 31, 101 and 104 (C31, C101, C104), histidines 86 and 90 (H86 and H90) and glutamate 114 (E114) of SORSa were chosen as the target amino acid residues for site-specific mutagenesis. The wild type and six mutant SORs were expressed in E. coli BL21, purified and confirmed by SDS-PAGE and Western blotting analysis. Enzyme activity determination revealed that the active sites of SORSa were identical with the archaea and Gram-negative bacteria SORs reported. Replacement of any cysteine residues reduced SOR activity by 53-100%, while the mutants of H86A, H90A and E114A lost their enzyme activities largely, only remaining 20%, 19% and 32% activity of the wild type SOR respectively. This study will enrich our awareness for active sites of SOR in a Gram-positive bacterium.

  3. A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

    SciTech Connect

    Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla; Peterson, Scott N.; Caffrey, Michael; Anderson, Wayne F.; Lavie, Arnon

    2012-04-18

    Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.

  4. Designing a Highly Active Metal-Free Oxygen Reduction Catalyst in Membrane Electrode Assemblies for Alkaline Fuel Cells: Effects of Pore Size and Doping-Site Position.

    PubMed

    Lee, Seonggyu; Choun, Myounghoon; Ye, Youngjin; Lee, Jaeyoung; Mun, Yeongdong; Kang, Eunae; Hwang, Jongkook; Lee, Young-Ho; Shin, Chae-Ho; Moon, Seung-Hyeon; Kim, Soo-Kil; Lee, Eunsung; Lee, Jinwoo

    2015-08-03

    To promote the oxygen reduction reaction of metal-free catalysts, the introduction of porous structure is considered as a desirable approach because the structure can enhance mass transport and host many catalytic active sites. However, most of the previous studies reported only half-cell characterization; therefore, studies on membrane electrode assembly (MEA) are still insufficient. Furthermore, the effect of doping-site position in the structure has not been investigated. Here, we report the synthesis of highly active metal-free catalysts in MEAs by controlling pore size and doping-site position. Both influence the accessibility of reactants to doping sites, which affects utilization of doping sites and mass-transport properties. Finally, an N,P-codoped ordered mesoporous carbon with a large pore size and precisely controlled doping-site position showed a remarkable on-set potential and produced 70% of the maximum power density obtained using Pt/C.

  5. Positive regulation of the beta-galactosidase gene from Kluyveromyces lactis is mediated by an upstream activation site that shows homology to the GAL upstream activation site of Saccharomyces cerevisiae.

    PubMed Central

    Ruzzi, M; Breunig, K D; Ficca, A G; Hollenberg, C P

    1987-01-01

    In contrast to the Escherichia coli lac operon, the yeast beta-galactosidase gene is positively regulated. In the 5'-noncoding region of the Kluyveromyces lactis LAC4 gene, we mapped an upstream activation site (UAS) that is required for induction. This sequence, located between positions -435 and -326 from the start of translation, functions irrespective of its orientation and can confer lactose regulation to the heterologous CYC1 promoter. It is composed of at least two subsequences that must act in concert. One of these subsequences showed a strong homology to the UAS consensus sequence of the Saccharomyces cerevisiae GAL genes (E. Giniger, S. M. Varnum, and M. Ptashne, Cell 40:767-774, 1985). We propose that this region of homology located at about position -426 is a binding site for the product of the regulatory gene LAC9 which probably induces transcription of the LAC4 gene in a manner analogous to that of the GAL4 protein. PMID:3104772

  6. Enhancer-like activity of A1gR1-binding site in alginate gene activation: positional, orientational, and sequence specificity.

    PubMed Central

    Fujiwara, S; Zielinski, N A; Chakrabarty, A M

    1993-01-01

    Significant activation of promoters of alginate genes such as algD or algC occurs in mucoid Pseudomonas aeruginosa during its proliferation in the lungs of cystic fibrosis patients. These promoters have been shown to be responsive to environmental signals such as high osmolarity. The signaling is mediated by a so-called two-component signal transduction system, in which a soluble protein, AlgR2, undergoes autophosphorylation and transfers the phosphate to a DNA-binding response regulator protein, AlgR1. The phosphorylated form of AlgR1 has a high affinity for binding at upstream sequences of both the algC and algD promoters. Two AlgR1-binding sites (ABS) have been reported upstream of the algC gene. One of the two ABSs (algC-ABS1, located at -94 to -81) is critical for the algC activation process, while the second ABS (algC-ABS2, located at +161 to +174) is only weakly active. We now report the presence of a third ABS within the structural gene of algC, and this ABS (algC-ABS3) is also important for algC promoter activation. algC-ABS1 can be replaced functionally by algC-ABS2, algD-ABS1, or algD-ABS2 and somewhat weakly by algD-ABS3. Introduction of a half-integral turn in the DNA helix between the algC site of transcription initiation and algC-ABS1 allowed only slight reduction of promoter activity, suggesting that the binding site could be appreciably functional even when present in the opposite face of the helix. Activation of the algC promoter is independent of the relative location (upstream or downstream of the mRNA start site), the number of copies, or the orientation of algC-ABS1, suggesting that it behaves like a eukaryotic enhancer element in promoting transcription from the algC promoter. Images PMID:8366031

  7. List 9 - Active CERCLIS Sites:

    EPA Pesticide Factsheets

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

  8. HPLC-based activity profiling: discovery of piperine as a positive GABA(A) receptor modulator targeting a benzodiazepine-independent binding site.

    PubMed

    Zaugg, Janine; Baburin, Igor; Strommer, Barbara; Kim, Hyun-Jung; Hering, Steffen; Hamburger, Matthias

    2010-02-26

    A plant extract library was screened for GABA(A) receptor activity making use of a two-microelectrode voltage clamp assay on Xenopus laevis oocytes. An ethyl acetate extract of black pepper fruits [Piper nigrum L. (Piperaceae) 100 microg/mL] potentiated GABA-induced chloride currents through GABA(A) receptors (composed of alpha(1), beta(2), and gamma(2S) subunits) by 169.1 +/- 2.4%. With the aid of an HPLC-based activity profiling approach, piperine (5) was identified as the main active compound, together with 12 structurally related less active or inactive piperamides (1-4, 6-13). Identification was achieved by on-line high-resolution mass spectrometry and off-line microprobe 1D and 2D NMR spectroscopy, using only milligram amounts of extract. Compound 5 induced a maximum potentiation of the chloride currents by 301.9 +/- 26.5% with an EC(50) of 52.4 +/- 9.4 microM. A comparison of the modulatory activity of 5 and other naturally occurring piperamides enabled insights into structural features critical for GABA(A) receptor modulation. The stimulation of chloride currents through GABA(A) receptors by compound 5 was not antagonized by flumazenil (10 microM). These data show that piperine (5) represents a new scaffold of positive allosteric GABA(A) receptor modulators targeting a benzodiazepine-independent binding site.

  9. HPLC-Based Activity Profiling: Discovery of Piperine as a Positive GABAA Receptor Modulator Targeting a Benzodiazepine-Independent Binding Site

    PubMed Central

    Zaugg, Janine; Baburin, Igor; Strommer, Barbara; Kim, Hyun-Jung; Hering, Steffen; Hamburger, Matthias

    2011-01-01

    A plant extract library was screened for GABAA receptor activity making use of a two-microelectrode voltage clamp assay on Xenopus laevis oocytes. An ethyl acetate extract of black pepper fruits [Piper nigrum L. (Piperaceae) 100 μg/mL] potentiated GABA-induced chloride currents through GABAA receptors (composed of α1, β2, and γ2S subunits) by 169.1 ± 2.4%. With the aid of an HPLC-based activity profiling approach, piperine (5) was identified as the main active compound, together with 12 structurally related less active or inactive piperamides (1–4, 6–13). Identification was achieved by on-line high-resolution mass spectrometry and off-line microprobe 1D and 2D NMR spectroscopy, using only milligram amounts of extract. Compound 5 induced a maximum potentiation of the chloride currents by 301.9 ± 26.5% with an EC50 of 52.4 ± 9.4 μM. A comparison of the modulatory activity of 5 and other naturally occurring piperamides enabled insights into structural features critical for GABAA receptor modulation. The stimulation of chloride currents through GABAA receptors by compound 5 was not antagonized by flumazenil (10 μM). These data show that piperine (5) represents a new scaffold of positive allosteric GABAA receptor modulators targeting a benzodiazepine-independent binding site. PMID:20085307

  10. Use of the parallax-quench method to determine the position of the active-site loop of cholesterol oxidase in lipid bilayers.

    PubMed

    Chen, X; Wolfgang, D E; Sampson, N S

    2000-11-07

    To elucidate the cholesterol oxidase-membrane bilayer interaction, a cysteine was introduced into the active site lid at position-81 using the Brevibacterium enzyme. To eliminate the possibility of labeling native cysteine, the single cysteine in the wild-type enzyme was mutated to a serine without any change in activity. The loop-cysteine mutant was then labeled with acrylodan, an environment-sensitive fluorescence probe. The fluorescence increased and blue-shifted upon binding to lipid vesicles, consistent with a change into a more hydrophobic, i.e., lipid, environment. This acrylodan-labeled cholesterol oxidase was used to explore the pH, ionic strength, and headgroup dependence of binding. Between pH 6 and 10, there was no significant change in binding affinity. Incorporation of anionic lipids (phosphatidylserine) into the vesicles did not increase the binding affinity nor did altering the ionic strength. These experiments suggested that the interactions are primarily driven by hydrophobic effects not ionic effects. Using vesicles doped with either 5-doxyl phosphatidylcholine, 10-doxyl phosphatidylcholine, or phosphatidyl-tempocholine, quenching of acrylodan fluorescence was observed upon binding. Using the parallax method of London [Chattopadhyay, A., and London, E. (1987) Biochemistry 26, 39-45], the acrylodan ring is calculated to be 8.1 +/- 2.5 A from the center of the lipid bilayer. Modeling the acrylodan-cysteine residue as an extended chain suggests that the backbone of the loop does not penetrate into the lipid bilayer but interacts with the headgroups, i.e., the choline. These results demonstrate that cholesterol oxidase interacts directly with the lipid bilayer and sits on the surface of the membrane.

  11. The Zn2 Position in Metallo-beta-Lactamases is Critical for Activity: A Study on Chimeric Metal Sites on a Conserved Protein Scaffold

    SciTech Connect

    Gonzalez,J.; Medrano Martin, F.; Costello, A.; Tierney, D.; Vila, A.

    2007-01-01

    Metallo-?-lactamases (M?Ls) are bacterial Zn(II)-dependent hydrolases that confer broad-spectrum resistance to ?-lactam antibiotics. These enzymes can be subdivided into three subclasses (B1, B2 and B3) that differ in their metal binding sites and their characteristic tertiary structure. To date there are no clinically useful pan-M?L inhibitors available, mainly due to the unawareness of key catalytic features common to all M?L brands. Here we have designed, expressed and characterized two double mutants of BcII, a di-Zn(II) B1-M?L from Bacillus cereus, namely BcII-R121H/C221D (BcII-HD) and BcII-R121H/C221S (BcII-HS). These mutants display modified environments at the so-called Zn2 site or DCH site, reproducing the metal coordination environments of structurally related metallohydrolases. Through a combination of structural and functional studies, we found that BcII-HD is an impaired ?-lactamase even as a di-Zn(II) enzyme, whereas BcII-HS exhibits the ability to exist as mono or di-Zn(II) species in solution, with different catalytic performances. We show that these effects result from an altered position of Zn2, which is incapable of providing a productive interaction with the substrate ?-lactam ring. These results indicate that the position of Zn2 is essential for a productive substrate binding and hydrolysis.

  12. Exhaustive mutagenesis of six secondary active-site residues in Escherichia coli chorismate mutase shows the importance of hydrophobic side chains and a helix N-capping position for stability and catalysis.

    PubMed

    Lassila, Jonathan Kyle; Keeffe, Jennifer R; Kast, Peter; Mayo, Stephen L

    2007-06-12

    Secondary active-site residues in enzymes, including hydrophobic amino acids, may contribute to catalysis through critical interactions that position the reacting molecule, organize hydrogen-bonding residues, and define the electrostatic environment of the active site. To ascertain the tolerance of an important model enzyme to mutation of active-site residues that do not directly hydrogen bond with the reacting molecule, all 19 possible amino acid substitutions were investigated in six positions of the engineered chorismate mutase domain of the Escherichia coli chorismate mutase-prephenate dehydratase. The six secondary active-site residues were selected to clarify results of a previous test of computational enzyme design procedures. Five of the positions encode hydrophobic side chains in the wild-type enzyme, and one forms a helix N-capping interaction as well as a salt bridge with a catalytically essential residue. Each mutant was evaluated for its ability to complement an auxotrophic chorismate mutase deletion strain. Kinetic parameters and thermal stabilities were measured for variants with in vivo activity. Altogether, we find that the enzyme tolerated 34% of the 114 possible substitutions, with a few mutations leading to increases in the catalytic efficiency of the enzyme. The results show the importance of secondary amino acid residues in determining enzymatic activity, and they point to strengths and weaknesses in current computational enzyme design procedures.

  13. Positive Surface over Positive Scattered Data Sites.

    PubMed

    Ibraheem, Farheen; Hussain, Malik Zawwar; Bhatti, Akhlaq Ahmad

    2015-01-01

    The aim of this paper is to develop a local positivity preserving scheme when the data amassed from different sources is positioned at sparse points. The proposed algorithm first triangulates the irregular data using Delauny triangulation method, therewith interpolates each boundary and radial curve of the triangle by C¹ rational trigonometric cubic function. Half of the parameters in the description of the interpolant are constrained to keep up the positive shape of data while the remaining half are set free for users' requirement. Orthogonality of trigonometric function assures much smoother surface as compared to polynomial functions. The proposed scheme can be of great use in areas of surface reconstruction and deformation, signal processing, CAD/CAM design, solving differential equations, and image restoration.

  14. Positive cooperative regulation of double binding sites for human acetylcholinesterase.

    PubMed

    Liu, Hao; Ye, Wei; Chen, Hai-Feng

    2016-10-25

    Acetylcholinesterase is a potent enzyme that regulates neurotransmission by rapidly hydrolyzing the neurotransmitter acetylcholine in synapses of the nervous system. As drug target of anti-AD, it has catalytic and peripheral anionic sites. However, the regulation relation between these two sites is unclear. Therefore, we constructed dynamics fluctuation network based on all-atom molecular dynamics simulations to reveal the regulation mechanism. The results suggest that the correlation network in double-site system (hAChE/TZ5) is distinctly different from that in the free state and single-site systems (hAChE/huprine and hAChE/1YL). The community network analysis indicates that the information freely transfers from the peripheral anionic site to the catalytic active site in hAChE/TZ5. Furthermore, the binding free energy between the inhibitor and hAChE for hAChE/TZ5 is significantly lower than of either hAChE/huprine or hAChE/1YL. Thus, a hypothesis of 'positive cooperative regulation' is proposed for the regulation of double binding sites and further confirmed by the weakening and mutation community analyses. Finally, one possible cooperative regulation pathway of W86-TZ5-W286 was identified based on the shortest path algorithm and was confirmed by the network perturbation analysis. Interestingly, the regulation pathway for single-site systems is significantly different from that of dual-site system. The process targeting on the shortest pathway can better regulate the hydrolyzing the neurotransmitter acetylcholine and significantly inhibit the aggregation of Aβ amyloid.

  15. Hormone activation induces nucleosome positioning in vivo

    PubMed Central

    Belikov, Sergey; Gelius, Birgitta; Almouzni, Geneviève; Wrange, Örjan

    2000-01-01

    The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone- and receptor-dependent activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the transition in chromatin structure following hormone activation. This revealed two novel findings: hormone activation led to the establishment of specific translational positioning of nucleosomes despite the lack of significant positioning in the inactive state; and, in the active promoter, a subnucleosomal particle encompassing the glucocorticoid receptor (GR)-binding region was detected. The presence of only a single GR-binding site was sufficient for the structural transition to occur. Both basal promoter elements and ongoing transcription were dispensable. These data reveal a stepwise process in the transcriptional activation by glucocorticoid hormone. PMID:10698943

  16. Activated levels of rRNA synthesis in fission yeast are driven by an intergenic rDNA region positioned over 2500 nucleotides upstream of the initiation site.

    PubMed Central

    Liu, Z; Zhao, A; Chen, L; Pape, L

    1997-01-01

    RNA polymerase I-catalyzed synthesis of the Schizosaccharomyces pombe approximately 37S pre-rRNAs was shown to be sensitive to regulatory sequences located several kilobases upstream of the initiation site for the rRNA gene. An in vitro transcription system for RNA polymerase I-catalyzed RNA synthesis was established that supports correct and activated transcription from templates bearing a full S. pombe rRNA gene promoter. A 780 bp region starting at -2560 significantly stimulates transcription of ac is-located rDNA promoter and competes with an rDNA promoter in trans, thus displaying some of the activities of rDNA transcriptional enhancers in vitro. Deletion of a 30 bp enhancer-homologous domain in this 780 bp far upstream region blocked its cis-stimulatory effect. The sequence of the S. pombe 3.5 kb intergenic spacer was determined and its organization differs from that of vertebrate, Drosophila, Acanthamoeba and plant intergenic rDNA spacers: it does not contain multiple, imperfect copies of the rRNA gene promoter nor repetitive elements of 140 bp, as are found in vertebrate rDNA enhancers. PMID:9016610

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

    PubMed

    Mikhaĭlik, I V; Verevka, S V

    1999-01-01

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

  18. 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.

  19. Positively selected sites in cetacean myoglobins contribute to protein stability.

    PubMed

    Dasmeh, Pouria; Serohijos, Adrian W R; Kepp, Kasper P; Shakhnovich, Eugene I

    2013-01-01

    Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10-20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the O2-binding affinity of Mbs is not significantly different among mammals (with typical oxygenation constants of ∼0.8-1.2 µM(-1)), folding stabilities of cetacean Mbs are ∼2-4 kcal/mol higher than for terrestrial Mbs. Using ancestral sequence reconstruction, maximum likelihood and bayesian tests to describe the evolution of cetacean Mbs, and experimentally calibrated computation of stability effects of mutations, we observe accelerated evolution in cetaceans and identify seven positively selected sites in Mb. Overall, these sites contribute to Mb stabilization with a conditional probability of 0.8. We observe a correlation between Mb folding stability and protein abundance, suggesting that a selection pressure for stability acts proportionally to higher expression. We also identify a major divergence event leading to the common ancestor of whales, during which major stabilization occurred. Most of the positively selected sites that occur later act against other destabilizing mutations to maintain stability across the clade, except for the shallow divers, where late stability relaxation occurs, probably due to the shorter aerobic dive limits of these species. The three main positively selected sites 66, 5, and 35 undergo changes that favor hydrophobic folding, structural integrity, and intra-helical hydrogen bonds.

  20. Private well/spring position paper, Rifle, Colorado, sites

    SciTech Connect

    1995-05-01

    This report describes the hydrogeochemical relationship between the New and Old Rifle processing sites and 15 domestic wells in their vicinity. The relationship of the domestic wells to the Old and New Rifle tailings sites requires clarification due to recent advances in understanding of Rifle site conceptual models. In order to form a bridge from the Rifle remedial action plan (RAP) and the recent baseline risk assessment to this position paper, several issues require discussion. First, through analysis of long-term ground water level data, the hydraulic gradient between the former tailings and private wells and springs was assessed. Second, in the Rifle RAP there was not a strong emphasis placed on describing regional influences on water quality in the vicinity of the processing sites. This document uses available information coupled with theory of regional ground water flow to describe regional flow systems north of Rifle. Third, the definition of background water quality from the RAP has been refined in several ways. Also, for the recent baseline risk assessment, all alluvial wells used to define background for the sites were located east of Old Rifle. In the RAP, alluvial background wells were also placed between the sites (downgradient of Old Rifle). Two additional wells were installed for the recent baseline risk assessment upgradient of Old Rifle which verified that several of the older wells (RFO-01-0597 and -0598) were in locations representative of background.

  1. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals

    PubMed Central

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)—short nucleotide sequences flanking introns—are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints. PMID:26642327

  2. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals.

    PubMed

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)--short nucleotide sequences flanking introns--are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints.

  3. Autonomous site selection and instrument positioning for sample acquisition

    NASA Astrophysics Data System (ADS)

    Shaw, A.; Barnes, D.; Pugh, S.

    The European Space Agency Aurora Exploration Program aims to establish a European long-term programme for the exploration of Space, culminating in a human mission to space in the 2030 timeframe. Two flagship missions, namely Mars Sample Return and ExoMars, have been proposed as recognised steps along the way. The Exomars Rover is the first of these flagship missions and includes a rover carrying the Pasteur Payload, a mobile exobiology instrumentation package, and the Beagle 2 arm. The primary objective is the search for evidence of past or present life on mars, but the payload will also study the evolution of the planet and the atmosphere, look for evidence of seismological activity and survey the environment in preparation for future missions. The operation of rovers in unknown environments is complicated, and requires large resources not only on the planet but also in ground based operations. Currently, this can be very labour intensive, and costly, if large teams of scientists and engineers are required to assess mission progress, plan mission scenarios, and construct a sequence of events or goals for uplink. Furthermore, the constraints in communication imposed by the time delay involved over such large distances, and line-of-sight required, make autonomy paramount to mission success, affording the ability to operate in the event of communications outages and be opportunistic with respect to scientific discovery. As part of this drive to reduce mission costs and increase autonomy the Space Robotics group at the University of Wales, Aberystwyth is researching methods of autonomous site selection and instrument positioning, directly applicable to the ExoMars mission. The site selection technique used builds on the geometric reasoning algorithms used previously for localisation and navigation [Shaw 03]. It is proposed that a digital elevation model (DEM) of the local surface, generated during traverse and without interaction from ground based operators, can be

  4. Normal Modes Expose Active Sites in Enzymes

    PubMed Central

    Glantz-Gashai, Yitav; Samson, Abraham O.

    2016-01-01

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

  5. Position Statement on Active Outdoor Play

    PubMed Central

    Tremblay, Mark S.; Gray, Casey; Babcock, Shawna; Barnes, Joel; Costas Bradstreet, Christa; Carr, Dawn; Chabot, Guylaine; Choquette, Louise; Chorney, David; Collyer, Cam; Herrington, Susan; Janson, Katherine; Janssen, Ian; Larouche, Richard; Pickett, William; Power, Marlene; Sandseter, Ellen Beate Hansen; Simon, Brenda; Brussoni, Mariana

    2015-01-01

    A diverse, cross-sectorial group of partners, stakeholders and researchers, collaborated to develop an evidence-informed Position Statement on active outdoor play for children aged 3–12 years. The Position Statement was created in response to practitioner, academic, legal, insurance and public debate, dialogue and disagreement on the relative benefits and harms of active (including risky) outdoor play. The Position Statement development process was informed by two systematic reviews, a critical appraisal of the current literature and existing position statements, engagement of research experts (N = 9) and cross-sectorial individuals/organizations (N = 17), and an extensive stakeholder consultation process (N = 1908). More than 95% of the stakeholders consulted strongly agreed or somewhat agreed with the Position Statement; 14/17 participating individuals/organizations endorsed it; and over 1000 additional individuals and organizations requested their name be listed as a supporter. The final Position Statement on Active Outdoor Play states: “Access to active play in nature and outdoors—with its risks— is essential for healthy child development. We recommend increasing children’s opportunities for self-directed play outdoors in all settings—at home, at school, in child care, the community and nature.” The full Position Statement provides context for the statement, evidence supporting it, and a series of recommendations to increase active outdoor play opportunities to promote healthy child development. PMID:26062040

  6. Position Statement on Active Outdoor Play.

    PubMed

    Tremblay, Mark S; Gray, Casey; Babcock, Shawna; Barnes, Joel; Bradstreet, Christa Costas; Carr, Dawn; Chabot, Guylaine; Choquette, Louise; Chorney, David; Collyer, Cam; Herrington, Susan; Janson, Katherine; Janssen, Ian; Larouche, Richard; Pickett, William; Power, Marlene; Sandseter, Ellen Beate Hansen; Simon, Brenda; Brussoni, Mariana

    2015-06-08

    A diverse, cross-sectorial group of partners, stakeholders and researchers, collaborated to develop an evidence-informed Position Statement on active outdoor play for children aged 3-12 years. The Position Statement was created in response to practitioner, academic, legal, insurance and public debate, dialogue and disagreement on the relative benefits and harms of active (including risky) outdoor play. The Position Statement development process was informed by two systematic reviews, a critical appraisal of the current literature and existing position statements, engagement of research experts (N=9) and cross-sectorial individuals/organizations (N=17), and an extensive stakeholder consultation process (N=1908). More than 95% of the stakeholders consulted strongly agreed or somewhat agreed with the Position Statement; 14/17 participating individuals/organizations endorsed it; and over 1000 additional individuals and organizations requested their name be listed as a supporter. The final Position Statement on Active Outdoor Play states: "Access to active play in nature and outdoors--with its risks--is essential for healthy child development. We recommend increasing children's opportunities for self-directed play outdoors in all settings--at home, at school, in child care, the community and nature." The full Position Statement provides context for the statement, evidence supporting it, and a series of recommendations to increase active outdoor play opportunities to promote healthy child development.

  7. Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation.

    PubMed Central

    Dankort, D L; Wang, Z; Blackmore, V; Moran, M F; Muller, W J

    1997-01-01

    A number of cytoplasmic signaling molecules are thought to mediate mitogenic signaling from the activated Neu receptor tyrosine kinase through binding specific phosphotyrosine residues located within the intracellular portion of Neu/c-ErbB-2. An activated neu oncogene containing tyrosine-to-phenylalanine substitutions at each of the known autophosphorylation sites was generated and assessed for its specific transforming potential in Rat1 and NIH 3T3 fibroblasts. Mutation of these sites resulted in a dramatic impairment of the transforming potential of neu. To assess the role of these tyrosine phosphorylation sites in cellular transformation, the transforming potential of a series of mutants in which individual tyrosine residues were restored to this transformation-debilitated neu mutant was evaluated. Reversion of any one of four mutated sites to tyrosine residues restored wild-type transforming activity. While each of these transforming mutants displayed Ras-dependent signaling, the transforming activity of two of these mutants was correlated with their ability to bind either the GRB2 or SHC adapter molecules that couple receptor tyrosine kinases to the Ras signaling pathway. By contrast, restoration of a tyrosine residue located at position 1028 completely suppressed the basal transforming activity of this mutated neu molecule or other transforming neu molecules which possessed single tyrosine residues. These data argue that the transforming potential of activated neu is mediated both by positive and negative regulatory tyrosine phosphorylation sites. PMID:9271418

  8. Discursive Positionings and Emotions in Modelling Activities

    ERIC Educational Resources Information Center

    Daher, Wajeeh

    2015-01-01

    Mathematical modelling is suggested as an activity through which students engage in meaningful mathematics. In the current research, the modelling activity of a group of four seventh-grade students was analysed using the discursive analysis framework. The research findings show that the positionings and emotions of the group members during their…

  9. Discursive positionings and emotions in modelling activities

    NASA Astrophysics Data System (ADS)

    Daher, Wajeeh

    2015-11-01

    Mathematical modelling is suggested as an activity through which students engage in meaningful mathematics. In the current research, the modelling activity of a group of four seventh-grade students was analysed using the discursive analysis framework. The research findings show that the positionings and emotions of the group members during their participation in the modelling activity changed as the activity proceeded. Overall, it can be said that three of the four group members acted as insiders, while the fourth acted as an outsider, and only, towards the end of the group's work on the activity, he acted as an insider. Moreover, the research findings point at four factors that affected the group members' positionings and emotions during the modelling activity: the member's characteristics, the member's history of learning experiences, the activity characteristics and the modelling phases. Furthermore, the different positionings of the group members in the different modelling phases were accompanied by different emotions experienced by them, where being an insider and a collaborator resulted in positive emotions, while being an outsider resulted in negative emotions.

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

    PubMed

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

    2012-01-04

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

  11. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

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

    2012-01-01

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

  12. Validated ligand mapping of ACE active site

    NASA Astrophysics Data System (ADS)

    Kuster, Daniel J.; Marshall, Garland R.

    2005-08-01

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

  13. Toward Understanding the Catalytic Mechanism of Human Paraoxonase 1: Site-Specific Mutagenesis at Position 192

    PubMed Central

    Aggarwal, Geetika; Prajapati, Rameshwar; Tripathy, Rajan K.; Bajaj, Priyanka; Iyengar, A. R. Satvik; Sangamwar, Abhay T.; Pande, Abhay H.

    2016-01-01

    Human paraoxonase 1 (h-PON1) is a serum enzyme that can hydrolyze a variety of substrates. The enzyme exhibits anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial and organophosphate-hydrolyzing activities. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against a variety conditions in human. However, the crystal structure of h-PON1 is not solved and the molecular details of how the enzyme hydrolyzes different substrates are not clear yet. Understanding the catalytic mechanism(s) of h-PON1 is important in developing the enzyme for therapeutic use. Literature suggests that R/Q polymorphism at position 192 in h-PON1 dramatically modulates the substrate specificity of the enzyme. In order to understand the role of the amino acid residue at position 192 of h-PON1 in its various hydrolytic activities, site-specific mutagenesis at position 192 was done in this study. The mutant enzymes were produced using Escherichia coli expression system and their hydrolytic activities were compared against a panel of substrates. Molecular dynamics simulation studies were employed on selected recombinant h-PON1 (rh-PON1) mutants to understand the effect of amino acid substitutions at position 192 on the structural features of the active site of the enzyme. Our results suggest that, depending on the type of substrate, presence of a particular amino acid residue at position 192 differentially alters the micro-environment of the active site of the enzyme resulting in the engagement of different subsets of amino acid residues in the binding and the processing of substrates. The result advances our understanding of the catalytic mechanism of h-PON1. PMID:26829396

  14. Mutation of Glycosylation Sites in BST-2 Leads to Its Accumulation at Intracellular CD63-Positive Vesicles without Affecting Its Antiviral Activity against Multivesicular Body-Targeted HIV-1 and Hepatitis B Virus.

    PubMed

    Han, Zhu; Lv, Mingyu; Shi, Ying; Yu, Jinghua; Niu, Junqi; Yu, Xiao-Fang; Zhang, Wenyan

    2016-02-29

    BST-2/tetherin blocks the release of various enveloped viruses including HIV-1 with a "physical tethering" model. The detailed contribution of N-linked glycosylation to this model is controversial. Here, we confirmed that mutation of glycosylation sites exerted an effect of post-translational mis-trafficking, leading to an accumulation of BST-2 at intracellular CD63-positive vesicles. BST-2 with this phenotype potently inhibited the release of multivesicular body-targeted HIV-1 and hepatitis B virus, without affecting the co-localization of BST-2 with EEA1 and LAMP1. These results suggest that N-linked glycosylation of human BST-2 is dispensable for intracellular virion retention and imply that this recently discovered intracellular tethering function may be evolutionarily distinguished from the canonical antiviral function of BST-2 by tethering nascent virions at the cell surface.

  15. Signal-regulator interactions. Genetic analysis of the effector binding site of xylS, the benzoate-activated positive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon.

    PubMed

    Ramos, J L; Michan, C; Rojo, F; Dwyer, D; Timmis, K

    1990-01-20

    This study reports a genetic analysis of the interactions between a positive regulator of gene expression and its effector molecules. Transcription of the TOL plasmid meta-cleavage pathway operon is specifically stimulated by the XylS protein positive regulator either through activation of this regulator by benzoate effectors or through its hyperproduction. One xylS mutant that exhibits constitutive expression of the operon promoter has been characterized, together with six mutants encoding altered XylS proteins that recognize as effectors benzoate analogues that are non-effectors for the XylS wild-type protein. The changes in two mutant regulators are located at the N-terminal end of the protein, within a putative beta-pleated domain. These mutant proteins exhibit a markedly increased affinity for normal benzoate effectors, with K's values fivefold to 60-fold lower than those of the wild-type XylS protein. They are additionally activated by new effectors having certain substituents at position 2, 3 and 4 of the aromatic ring. Two other mutant proteins recognize new effectors having substituents at position 4 and 5 of the aromatic ring, and contain mutations at their C-terminal end within a putative alpha-helix-rich domain. Three other mutations, one of which leads to constitutive expression from Pm, each result in an amino acid change in the central region of the regulator. These findings suggest but do not prove that the effector binding pocket of the XylS protein may be composed of two or more non-contiguous segments of its primary structure. The XylS protein exhibits homology with the AraC protein of Escherichia coli, a protein that stimulates transcription from ara promoters when it is activated by arabinose or benzoate. Mutations influencing effector activation of the XylS protein characterized in this study are all located in regions exhibiting a high degree of homology with the corresponding aligned sequence of AraC protein.

  16. Phylogenetic analysis and positive-selection site detecting of vascular endothelial growth factor family in vertebrates.

    PubMed

    He, Wenwu; Tang, Yanyan; Qi, Bin; Lu, Chuansen; Qin, Chao; Wei, Yunfei; Yi, Jiachao; Chen, Mingwu

    2014-02-10

    Vascular endothelial growth factor (VEGF), known to play an important role in vascular homeostasis, vascular integrity and angiogenesis, is little known about the evolutionary relationship of its five members especially the role of gene duplication and natural selection in the evolution of the VEGF family. In this study, seventy-five full-length cDNA sequences from 33 vertebrate species were extracted from the NCBI's GenBank, UniProt protein database and the Ensembl database. By phylogenetic analyses, we investigated the origin, conservation, and evolution of the VEGFs. Five VEGF family members in vertebrates might be formed by gene duplication. The inferred evolutionary transitions that separate members which belong to different gene clusters correlated with changes in functional properties. Selection analysis and protein structure analysis were combined to explain the relationship of the site-specific evolution in the vertebrate VEGF family. Eleven positive selection sites, one transmembrane region and the active sites were detected in this process.

  17. 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.

  18. 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.

  19. Promoter-proximal polyadenylation sites reduce transcription activity

    PubMed Central

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

    2012-01-01

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

  20. Spectroscopic studies of the active site of galactose oxidase

    SciTech Connect

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

    1995-07-19

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

  1. Potential sites of CFTR activation by tyrosine kinases

    PubMed Central

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

    2016-01-01

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

  2. Criteria for Formation of Active Personal Position of Schoolchildren

    ERIC Educational Resources Information Center

    Kunanbayeva, Magziya Sh.

    2016-01-01

    The article considers the problem and the importance of formation of the active personal position of schoolchildren. Active personal position is a complex concept, which includes the ability to a problem solution, the ability to work in a team, the ability to express his or her views. The formation of an active personal position at school is…

  3. 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.

  4. Comprehensive School Physical Activity Programs. Position Statement

    ERIC Educational Resources Information Center

    National Association for Sport and Physical Education, 2008

    2008-01-01

    The National Association for Sport and Physical Education (NASPE) recommends that all PK-12 schools implement a Comprehensive School Physical Activity Program. Schools play an important role in public health, and the physical, mental, and social benefits of regular physical activity for youth are well documented. Leading public health, medical,…

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

    SciTech Connect

    Cheng, K.C.

    1989-01-01

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

  6. ON-LINE TOOLS FOR PROPER VERTICAL POSITIONING OF VERTICAL SAMPLING INTERVALS DURING SITE ASSESSMENT

    EPA Science Inventory

    This presentation presents on-line tools for proper vertical positioning of vertical sampling intervals during site assessment. Proper vertical sample interval selection is critical for generate data on the vertical distribution of contamination. Without vertical delineation, th...

  7. 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.

  8. Estimating the relative position of risperidone primary binding site in Sera Albumins. Modeling from spectrofluorimetric data

    NASA Astrophysics Data System (ADS)

    Cortez, Celia Martins; Fragoso, Viviane Muniz S.; Silva, Dilson

    2014-10-01

    In this work, we used a mathematical model to study the interaction of risperidone with human and bovine serum albumins estimating the relative position of the primary binding site, based on the fluorescence quenching theory. Results have shown that the model was able to demonstrate that primary binding site for risperidone in HSA and BSA is very close to the position where is tryptophan 134 of BSA, possibly in domain 1B.

  9. Position specific variation in the rate of evolution intranscription factor binding sites

    SciTech Connect

    Moses, Alan M.; Chiang, Derek Y.; Kellis, Manolis; Lander, EricS.; Eisen, Michael B.

    2003-08-28

    The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Here we analyze the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikataeto study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artifacts of computational motif finding algorithms. As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative

  10. Fragile phagocytes: FMRP positively regulates engulfment activity.

    PubMed

    Logan, Mary A

    2017-03-06

    Defective immune system function is implicated in autism spectrum disorders, including Fragile X syndrome. In this issue, O'Connor et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201607093) demonstrate that phagocytic activity of systemic immune cells is compromised in a Drosophila melanogaster model of Fragile X, highlighting intriguing new mechanistic connections between FMRP, innate immunity, and abnormal development.

  11. Strong and widespread action of site-specific positive selection in the snake venom Kunitz/BPTI protein family

    PubMed Central

    Župunski, Vera; Kordiš, Dušan

    2016-01-01

    S1 family of serine peptidases is the largest family of peptidases. They are specifically inhibited by the Kunitz/BPTI inhibitors. Kunitz domain is characterized by the compact 3D structure with the most important inhibitory loops for the inhibition of S1 peptidases. In the present study we analysed the action of site-specific positive selection and its impact on the structurally and functionally important parts of the snake venom Kunitz/BPTI family of proteins. By using numerous models we demonstrated the presence of large numbers of site-specific positively selected sites that can reach between 30–50% of the Kunitz domain. The mapping of the positively selected sites on the 3D model of Kunitz/BPTI inhibitors has shown that these sites are located in the inhibitory loops 1 and 2, but also in the Kunitz scaffold. Amino acid replacements have been found exclusively on the surface, and the vast majority of replacements are causing the change of the charge. The consequence of these replacements is the change in the electrostatic potential on the surface of the Kunitz/BPTI proteins that may play an important role in the precise targeting of these inhibitors into the active site of S1 family of serine peptidases. PMID:27841308

  12. 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.

  13. Positive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase.

    PubMed

    Bury, Daniel; Dahmane, Ismahene; Derouaux, Adeline; Dumbre, Shrinivas; Herdewijn, Piet; Matagne, André; Breukink, Eefjan; Mueller-Seitz, Erika; Petz, Michael; Terrak, Mohammed

    2015-01-15

    The glycosyltransferases of family 51 (GT51) catalyze the polymerization of lipid II to form linear glycan chains, which, after cross linking by the transpeptidases, form the net-like peptidoglycan macromolecule. The essential function of the GT makes it an attractive antimicrobial target; therefore a better understanding of its function and its mechanism of interaction with substrates could help in the design and the development of new antibiotics. In this work, we have used a surface plasmon resonance Biacore(®) biosensor, based on an amine derivative of moenomycin A immobilized on a sensor chip surface, to investigate the mechanism of binding of substrate analogous inhibitors to the GT. Addition of increasing concentrations of moenomycin A to the Staphylococcus aureus MtgA led to reduced binding of the protein to the sensor chip as expected. Remarkably, in the presence of low concentrations of the most active disaccharide inhibitors, binding of MtgA to immobilized moenomycin A was found to increase; in contrast competition with moenomycin A occurred only at high concentrations. This finding suggests that at low concentrations, the lipid II analogs bind to the acceptor site and induce a cooperative binding of moenomycin A to the donor site. Our results constitute the first indication of the existence of a positive cooperativity between the acceptor and the donor sites of peptidoglycan GTs. In addition, our study indicates that a modification of two residues (L119N and F120S) within the hydrophobic region of MtgA can yield monodisperse forms of the protein with apparently no change in its secondary structure content, but this is at the expense of the enzyme function.

  14. Transcription Factor Binding Site Positioning in Yeast: Proximal Promoter Motifs Characterize TATA-Less Promoters

    PubMed Central

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of ‘proximal promoter motifs’ (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters. PMID:21931670

  15. Transcription factor binding site positioning in yeast: proximal promoter motifs characterize TATA-less promoters.

    PubMed

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 6 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.

  16. A Flexible Binding Site Architecture Provides New Insights into CcpA Global Regulation in Gram-Positive Bacteria

    PubMed Central

    Yang, Yunpeng; Zhang, Lu; Huang, He; Yang, Chen; Yang, Sheng

    2017-01-01

    ABSTRACT Catabolite control protein A (CcpA) is the master regulator in Gram-positive bacteria that mediates carbon catabolite repression (CCR) and carbon catabolite activation (CCA), two fundamental regulatory mechanisms that enable competitive advantages in carbon catabolism. It is generally regarded that CcpA exerts its regulatory role by binding to a typical 14- to 16-nucleotide (nt) consensus site that is called a catabolite response element (cre) within the target regions. However, here we report a previously unknown noncanonical flexible architecture of the CcpA-binding site in solventogenic clostridia, providing new mechanistic insights into catabolite regulation. This novel CcpA-binding site, named crevar, has a unique architecture that consists of two inverted repeats and an intervening spacer, all of which are variable in nucleotide composition and length, except for a 6-bp core palindromic sequence (TGTAAA/TTTACA). It was found that the length of the intervening spacer of crevar can affect CcpA binding affinity, and moreover, the core palindromic sequence of crevar is the key structure for regulation. Such a variable architecture of crevar shows potential importance for CcpA’s diverse and fine regulation. A total of 103 potential crevar sites were discovered in solventogenic Clostridium acetobutylicum, of which 42 sites were picked out for electrophoretic mobility shift assays (EMSAs), and 30 sites were confirmed to be bound by CcpA. These 30 crevar sites are associated with 27 genes involved in many important pathways. Also of significance, the crevar sites are found to be widespread and function in a great number of taxonomically different Gram-positive bacteria, including pathogens, suggesting their global role in Gram-positive bacteria. PMID:28119470

  17. 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

  18. Positive Darwinian selection at single amino acid sites conferring plant virus resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Explicit evaluation of the accuracy and power of Maximum Likelihood and Bayesian methods for detecting site-specific positive Darwinian selection presents a challenge because selective consequences of single amino acid changes are generally unknown. We exploit extensive molecular and functional cha...

  19. Tryptophan analogues. 1. Synthesis and antihypertensive activity of positional isomers.

    PubMed

    Safdy, M E; Kurchacova, E; Schut, R N; Vidrio, H; Hong, E

    1982-06-01

    A series of tryptophan analogues having the carboxyl function at the beta-position was synthesized and tested for antihypertensive activity. The 5-methoxy analogue 46 exhibited antihypertensive activity in the rat via the oral route and was much more potent than the normal tryptophan analogue. The methyl ester was found to be a critical structural feature for activity.

  20. Positive selection neighboring functionally essential sites and disease-implicated regions of mammalian reproductive proteins

    PubMed Central

    2010-01-01

    Background Reproductive proteins are central to the continuation of all mammalian species. The evolution of these proteins has been greatly influenced by environmental pressures induced by pathogens, rival sperm, sexual selection and sexual conflict. Positive selection has been demonstrated in many of these proteins with particular focus on primate lineages. However, the mammalia are a diverse group in terms of mating habits, population sizes and germ line generation times. We have examined the selective pressures at work on a number of novel reproductive proteins across a wide variety of mammalia. Results We show that selective pressures on reproductive proteins are highly varied. Of the 10 genes analyzed in detail, all contain signatures of positive selection either across specific sites or in specific lineages or a combination of both. Our analysis of SP56 and Col1a1 are entirely novel and the results show positively selected sites present in each gene. Our findings for the Col1a1 gene are suggestive of a link between positive selection and severe disease type. We find evidence in our dataset to suggest that interacting proteins are evolving in symphony: most likely to maintain interacting functionality. Conclusion Our in silico analyses show positively selected sites are occurring near catalytically important regions suggesting selective pressure to maximize efficient fertilization. In those cases where a mechanism of protein function is not fully understood, the sites presented here represent ideal candidates for mutational study. This work has highlighted the widespread rate heterogeneity in mutational rates across the mammalia and specifically has shown that the evolution of reproductive proteins is highly varied depending on the species and interacting partners. We have shown that positive selection and disease are closely linked in the Col1a1 gene. PMID:20149245

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

    SciTech Connect

    Ivanov, Ivaylo; Klien, Micheal

    2004-01-14

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

  2. Activities for Teaching Positive Psychology: A Guide for Instructors

    ERIC Educational Resources Information Center

    Froh, Jeffrey J., Ed.; Parks, Acacia C., Ed.

    2012-01-01

    Positive psychology is a rapidly expanding area of study that is of great interest to students at the graduate, undergraduate, and high school levels. But the field is so broad that teachers who want to cover all the bases when designing a positive psychology course may have difficulty locating and selecting materials. "Activities for Teaching…

  3. Laser optical disk position encoder with active heads

    NASA Technical Reports Server (NTRS)

    Osborne, Eric P.

    1991-01-01

    An angular position encoder that minimizes the effects of eccentricity and other misalignments between the disk and the read stations by employing heads with beam steering optics that actively track the disk in directions along the disk radius and normal to its surface is discussed. The device adapts features prevalent in optical disk technology to the application of angular position sensing.

  4. Positive selection sites in tertiary structure of Leguminosae chalcone isomerase 1.

    PubMed

    Wang, R K; Zhan, S F; Zhao, T J; Zhou, X L; Wang, C E

    2015-03-20

    Isoflavonoids and the related synthesis enzyme, chalcone isomerase 1 (CHI1), are unique in the Leguminosae, with diverse biological functions. Among the Leguminosae, the soybean is an important oil, protein crop, and model plant. In this study, we aimed to detect the generation pattern of Leguminosae CHI1. Genome-wide sequence analysis of CHI in 3 Leguminosae and 3 other closely related model plants was performed; the expression levels of soybean chalcone isomerases were also analyzed. By comparing positively selected sites and their protein structures, we retrieved the evolution patterns for Leguminosae CHI1. A total of 28 CHI and 7 FAP3 (CHI4) genes were identified and separated into 4 clades: CHI1, CHI2, CHI3, and FAP3. Soybean genes belonging to the same chalcone isomerase subfamily had similar expression patterns. CHI1, the unique chalcone isomerase subfamily in Leguminosae, showed signs of significant positive selection as well as special expression characteristics, indicating an accelerated evolution throughout its divergence. Eight sites were identified as undergoing positive selection with high confidence. When mapped onto the tertiary structure of CHI1, these 8 sites were observed surrounding the enzyme substrate only; some of them connected to the catalytic core of CHI. Thus, we inferred that the generation of Leguminosae CHI1 is dependent on the positively selected amino acids surrounding its catalytic substrate. In other words, the evolution of CHI1 was driven by specific selection or processing conditions within the substrate.

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

    SciTech Connect

    Hartman, F.C.

    1991-01-01

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

  6. 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.

  7. 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.

  8. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    2016-01-01

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

  11. Site-specific aflatoxin B sub 1 adduction of sequence-positioned nucleosome core particles

    SciTech Connect

    Moyer, R.A.

    1988-01-01

    The question of how the presence of nucleosomal packing of DNA modifies carcinogen interaction at specific sites cannot be answered by studies on whole chromatin or bulk nucleosomes because of the heterogeneity of DNA sequences in the particles. This problem was circumvented by constructing nucleosomes that are homogenous in DNA-histone contact points. A cloned DNA fragment, containing a sea urchin 5S gene which precisely positions a histone octamer was employed. By using {sup 32}P end-labeled DNA and genotoxins that allow cleavage at sites of attack, the frequency of adduction at every susceptible nucleotide can be determined on sequencing gels. The small methylating agent dimethyl sulfate (DMS) and the bulky alkylating agent afatoxin B{sub 1}-dichloride (AFB{sub 1}-Cl{sub 2}) were used to probe the influence of DNA-histone interactions on DNA alkylation patterns in sequence-positioned core particles.

  12. Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

    PubMed Central

    Zhang, Jiantao; Zhang, Zhenlu; Chukkapalli, Vineela; Nchoutmboube, Jules A.; Li, Jianhui; Randall, Glenn; Belov, George A.; Wang, Xiaofeng

    2016-01-01

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes. PMID:26858414

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

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

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

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

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

  15. Robotics at Savannah River site: activity report

    SciTech Connect

    Byrd, J.S.

    1984-09-01

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

  16. Promoting positive psychology using social networking sites: a study of new college entrants on Facebook.

    PubMed

    Chang, Shu-Man; Lin, Yung-Hsiu; Lin, Chi-Wei; Chang, Her-Kun; Chong, Ping Pete

    2014-04-29

    This study explores the potential of promoting college students' positive psychological development using popular online social networks. Online social networks have dramatically changed the ways college students manage their social relationships. Social network activities, such as checking Facebook posts dominates students' Internet time and has the potential to assist students' positive development. Positive psychology is a scientific study of how ordinary individuals can apply their strength effectively when facing objective difficulties and how this capability can be cultivated with certain approaches. A positive message delivery approach was designed for a group of new college entrants. A series of positive messages was edited by university counselors and delivered by students to their Facebook social groups. Responses from each posted positive messages were collected and analyzed by researchers. The responses indicated that: (1) relationships of individual engagement and social influence in this study can partially explain the observed student behavior; (2) using class-based social groups can promote a positive atmosphere to enhance strong-tie relationships in both the physical and virtual environments, and (3) promoting student's positive attitudes can substantially impact adolescents' future developments, and many positive attitudes can be cultivated by emotional events and social influence.

  17. Phosphorylation of human Jak3 at tyrosines 904 and 939 positively regulates its activity.

    PubMed

    Cheng, Hanyin; Ross, Jeremy A; Frost, Jeffrey A; Kirken, Robert A

    2008-04-01

    Janus tyrosine kinase 3 (Jak3) is essential for signaling by interleukin-2 (IL-2) family cytokines and proper immune function. Dysfunctional regulation of Jak3 may result in certain disease states. However, the molecular mechanisms governing Jak3 activation are not fully understood. In this study, we used a functional-proteomics approach to identify two novel tyrosine phosphorylation sites within Jak3, Y904 and Y939, which are conserved among Jak family proteins. By using phosphospecific antibodies, both residues were observed to be rapidly induced by stimulation of cells with IL-2 or other gammac cytokines. Mechanistic studies indicated that Y904 and Y939 regulate Jak3 activities. A phenylalanine substitution at either site greatly reduced Jak3 kinase activity in vitro and its ability to phosphorylate signal transducer and activator of transcription 5 (Stat5) in vivo, suggesting that phosphorylation of these previously unrecognized residues positively regulates Jak3 activity. Y904 and Y939 were required for optimal ATP usage by Jak3, while phosphorylation of Y939 preferentially promoted Stat5 activity in intact cells. Together, these findings demonstrate positive functional roles for two novel Jak3 phosphoregulatory sites which may be similarly important for other Jak family members. Identification of these sites also provides new therapeutic opportunities to modulate Jak3 function.

  18. Active sites of thioredoxin reductases: why selenoproteins?

    PubMed

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

    2003-10-28

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

  19. Global positioning system: a new opportunity in physical activity measurement.

    PubMed

    Maddison, Ralph; Ni Mhurchu, Cliona

    2009-11-04

    Accurate measurement of physical activity is a pre-requisite to monitor population physical activity levels and design effective interventions. Global Positioning System (GPS) technology offers potential to improve the measurement of physical activity. This paper 1) reviews the extant literature on the application of GPS to monitor human movement, with a particular emphasis on free-living physical activity, 2) discusses issues associated with GPS use, and 3) provides recommendations for future research. Overall findings show that GPS is a useful tool to augment our understanding of physical activity by providing the context (location) of the activity and used together with Geographical Information Systems can provide some insight into how people interact with the environment. However, no studies have shown that GPS alone is a reliable and valid measure of physical activity.

  20. Global positioning system: a new opportunity in physical activity measurement

    PubMed Central

    Maddison, Ralph; Ni Mhurchu, Cliona

    2009-01-01

    Accurate measurement of physical activity is a pre-requisite to monitor population physical activity levels and design effective interventions. Global Positioning System (GPS) technology offers potential to improve the measurement of physical activity. This paper 1) reviews the extant literature on the application of GPS to monitor human movement, with a particular emphasis on free-living physical activity, 2) discusses issues associated with GPS use, and 3) provides recommendations for future research. Overall findings show that GPS is a useful tool to augment our understanding of physical activity by providing the context (location) of the activity and used together with Geographical Information Systems can provide some insight into how people interact with the environment. However, no studies have shown that GPS alone is a reliable and valid measure of physical activity. PMID:19887012

  1. Division site positioning in bacteria: one size does not fit all

    PubMed Central

    Monahan, Leigh G.; Liew, Andrew T. F.; Bottomley, Amy L.; Harry, Elizabeth J.

    2014-01-01

    Spatial regulation of cell division in bacteria has been a focus of research for decades. It has been well studied in two model rod-shaped organisms, Escherichia coli and Bacillus subtilis, with the general belief that division site positioning occurs as a result of the combination of two negative regulatory systems, Min and nucleoid occlusion. These systems influence division by preventing the cytokinetic Z ring from forming anywhere other than midcell. However, evidence is accumulating for the existence of additional mechanisms that are involved in controlling Z ring positioning both in these organisms and in several other bacteria. In some cases the decision of where to divide is solved by variations on a common evolutionary theme, and in others completely different proteins and mechanisms are involved. Here we review the different ways bacteria solve the problem of finding the right place to divide. It appears that a one-size-fits-all model does not apply, and that individual species have adapted a division-site positioning mechanism that best suits their lifestyle, environmental niche and mode of growth to ensure equal partitioning of DNA for survival of the next generation. PMID:24550892

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

    EPA Pesticide Factsheets

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

  3. Condensing position-specific scoring matrixs by the Kidera factors for ligand-binding site prediction.

    PubMed

    Fang, Chun; Noguchi, Tamotsu; Yamana, Hayato

    2015-01-01

    Position-specific scoring matrix (PSSM) has been widely used for identifying protein functional sites. However, it is 20-dimentional and contains many redundant features. The Kidera factors were reported to contain information relating almost all physical properties of amino acids, but it requires appropriate weighting coefficients to express their properties. We developed a novel method, named as KSPSSMpred, which integrated PSSM and the Kidera Factors into a 10-dimensional matrix (KSPSSM) for ligand-binding site prediction. Flavin adenine dinucleotide (FAD) was chosen as a representative ligand for this study. When compared with five other feature-based methods on a benchmark dataset, KSPSSMpred performed the best. This study demonstrates that, KSPSSM is an effective feature extraction method which can enrich PSSM with information relating 188 physical properties of residues, and reduce 50% feature dimensions without losing information included in the PSSM.

  4. Physical Activity Is Positively Associated with Episodic Memory in Aging

    PubMed Central

    Hayes, Scott M.; Alosco, Michael L.; Hayes, Jasmeet P.; Cadden, Margaret; Peterson, Kristina M.; Allsup, Kelly; Forman, Daniel E.; Sperling, Reisa A.; Verfaellie, Mieke

    2016-01-01

    Aging is associated with performance reductions in executive function and episodic memory, although there is substantial individual variability in cognition among older adults. One factor that may be positively associated with cognition in aging is physical activity. To date, few studies have objectively assessed physical activity in young and older adults, and examined whether physical activity is differentially associated with cognition in aging. Young (n = 29, age 18–31 years) and older adults (n = 31, ages 55–82 years) completed standardized neuropsychological testing to assess executive function and episodic memory capacities. An experimental face-name relational memory task was administered to augment assessment of episodic memory. Physical activity (total step count and step rate) was objectively assessed using an accelerometer, and hierarchical regressions were used to evaluate relationships between cognition and physical activity. Older adults performed more poorly on tasks of executive function and episodic memory. Physical activity was positively associated with a composite measure of visual episodic memory and face-name memory accuracy in older adults. Physical activity associations with cognition were independent of sedentary behavior, which was negatively correlated with memory performance. Physical activity was not associated with cognitive performance in younger adults. Physical activity is positively associated with episodic memory performance in aging. The relationship appears to be strongest for face-name relational memory and visual episodic memory, likely attributable to the fact that these tasks make strong demands on the hippocampus. The results suggest that physical activity relates to cognition in older, but not younger adults. PMID:26581790

  5. MapZ beacons the division sites and positions FtsZ-rings in Streptococcus pneumoniae

    PubMed Central

    Zhao, Chao; Cluzel, Caroline; Lavergne, Jean-Pierre; Franz-Wachtel, Mirita; Macek, Boris; Combet, Christophe; Kuru, Erkin; VanNieuwenhze, Michael S.; Brun, Yves V.; Sherratt, David; Grangeasse, Christophe

    2014-01-01

    In every living organism, cell division requires accurate identification of the division site and placement of the division machinery. In bacteria, this process is traditionally considered to begin with the polymerization of the highly conserved tubulin-like protein FtsZ into a ring that locates precisely at midcell1. Over the last decades, several systems have been reported to regulate the spatiotemporal assembly and placement of the FtsZ-ring2-5. However, the human pathogen Streptococcus pneumoniae, as many other organisms, is devoid of these canonical systems and the mechanisms of positioning of the division machinery remain unknown4,6. Here we characterize a novel factor that locates at the division site before FtsZ and guides septum positioning in the pneumococcus. MapZ (Midcell Anchored Protein Z) forms ring structures at the cell equator and moves apart as the cell elongates, therefore behaving as a permanent beacon of division sites. MapZ then positions the FtsZ-ring through direct protein-protein interactions. MapZ-mediated control differs from previously described systems mostly based on negative regulation of FtsZ assembly. Further, MapZ is an endogenous target of the ser/thr-kinase StkP, which was recently shown to play a central role in cytokinesis and morphogenesis of the pneumococcus7-9. We show that both phosphorylated and non-phosphorylated forms of MapZ are required for proper Z-ring formation and dynamics. Altogether, this work uncovers a new mechanism for bacterial cell division that is regulated by phosphorylation and illustrates that nature has evolved a diversity of cell division mechanisms adapted to the different bacterial clades. PMID:25470041

  6. Draft position paper on knowledge management in space activities

    NASA Technical Reports Server (NTRS)

    Holm, Jeanne; Moura, Denis

    2003-01-01

    As other fields of industry, space activities are facing the challenge of Knowledge Management and the International Academy of Astronautics decided to settle in 2002 a Study Group to analyse the problem and issue general guidelines. This communication presents the draft position paper of this group in view to be discussed during the 2003 IAF Congress.

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

    PubMed

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

    1999-10-22

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

  8. DNaseI hypersensitive sites 1, 2 and 3 of the human beta-globin dominant control region direct position-independent expression.

    PubMed Central

    Fraser, P; Hurst, J; Collis, P; Grosveld, F

    1990-01-01

    The human beta-globin dominant control region (DCR) which flanks the multigene beta-globin locus directs high level, site of integration independent, copy number dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukemia (MEL) cells. We have assayed each of the individual DNaseI hypersensitive regions present in the full 15kb DCR for position independence and copy number dependence of a linked beta-globin gene in transgenic mice. The results show that at least three of the individual DNaseI hypersensitive site regions (sites 1, 2 and 3), though expressing at lower levels than the full DCR, are capable of position independent, copy number dependent expression. Site 2 alone directs the highest level of expression of the single site constructs, producing nearly 70% of the level of the full DCR. Sites 1 and 3 each provide 30% of the full activity. Deletion of either site 2 or 3 from the complete set significantly reduces the level of expression, but does not effect position independence or copy number dependence. This demonstrates that sites 2 and 3 are required for full expression and suggests that all the sites are required for the full expression of even a single gene from this multigene locus. Images PMID:2362805

  9. Relative position and pose measurement approach of specific operation site of space non-cooperative target

    NASA Astrophysics Data System (ADS)

    Chen, Feng; Zhou, Ying; Li, Ronghua; Huang, Jianming

    2015-12-01

    In order to achieve the rendezvous and capture of the space non-cooperative target, the relative position and pose measurement of non-cooperative target must be resolved. Since the marker is not installed into the non-cooperative target and there is no inter satellite link to transfer the information, so it is very difficult to measure the relative position and pose measurement of non-cooperative target. The solar array connecting frame of non-cooperative targets have their characters and are easy to capture, so the position and pose measurement of specific operation site of non-cooperative target based on stereo vision has been studied in this paper. The method composed of image acquiring, image filtering, edge detection, feature extraction and relative pose measurement. Finally, the relative position and attitude parameters of the solar wing connection were obtained and provided to the control system. The results of simulation and ground verification show that the algorithm is accurate and effective, and can satisfy the technical requirements of the on orbit operation. The measurement approach can be used for engineering implementation.

  10. Inorganic lead and calcium interact positively in activation of calmodulin.

    PubMed

    Kern, M; Wisniewski, M; Cabell, L; Audesirk, G

    2000-06-01

    Calmodulin is a ubiquitous calcium-binding protein that mediates many of the intracellular actions of Ca2+ ions. The calcium-binding sites of calmodulin consist of four EF-hand motifs; full activation of calmodulin normally occurs when all four sites are occupied by Ca2+. Inorganic lead (PY2+) has been shown to activate calmodulin at total lead concentrations similar to the concentrations of Ca2+ required for activation (Goldstein and Ar, 1983; Habermann et al., 1983), but the free Pb2+ concentrations required for calmodulin activation have not been determined. In addition, it is possible that activation may occur with different sites occupied by different divalent cations, for example Ca2+ and Pb2+. We investigated the ability of free Pb2+, alone or in combination with Ca2+, to activate calmodulin. In aqueous media, N-phenyl-1-naphthylamine (NPN) and 8-anilino-1-naphthalenesulfonate (ANS) show increased fluorescence when bound to hydrophobic regions of proteins. This increased fluorescence has been used to monitor the conformational change that occurs during calmodulin activation (LaPorte et al., 1980). In the presence of calmodulin, both Ca2+ and Pb2+ stimulated increased fluorescence of NPN and ANS. Threshold and EC50 free metal concentrations were approximately 100 nM and 450-500 nM, respectively, for Ca2+ and 100 pM and 400-550 pM, respectively, for Pb2+. Fluorescence was enhanced by combinations of low concentrations of free Ca2+ and Pb2+; for example, as little as 20 pM free Pb2+ enhanced fluorescence in combination with 200 nM free Ca2+. The activity of the PDE1 isoform of cyclic nucleotide phosphodiesterase is stimulated by Ca2+/calmodulin (Wang et al., 1990). In the presence of calmodulin, we found that Ca2+ and Pb2+ activated calmodulin-stimulated PDE activity, with threshold and EC50 free metal concentrations of approximately 200 nM and 1200 nM, respectively, for Ca2+ and 300 pM and 430 pM, respectively, for Pb2+. PDE activity was stimulated by

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

    PubMed Central

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

    2016-01-01

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

  12. Positioning.

    ERIC Educational Resources Information Center

    Conone, Ruth M.

    The key to positioning is the creation of a clear benefit image in the consumer's mind. One positioning strategy is creating in the prospect's mind a position that takes into consideration the company's or agency's strengths and weaknesses as well as those of its competitors. Another strategy is to gain entry into a position ladder owned by…

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

    PubMed

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

    2008-08-01

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

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

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

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

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

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

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

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

    PubMed

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

    2015-07-13

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

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

    PubMed

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

    2016-10-04

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

  18. Ultrastructure of the replication sites of positive-strand RNA viruses

    SciTech Connect

    Harak, Christian; Lohmann, Volker

    2015-05-15

    Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. In this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. - Highlights: • Positive strand RNA viruses induce massive membrane alterations. • Despite the great diversity, replication complexes share many similarities. • Host factors play a pivotal role in replication complex biogenesis. • Use of the same host factors by several viruses hints to similar functions.

  19. Positive Technology for Healthy Living and Active Ageing.

    PubMed

    Riva, Giuseppe; Gaggioli, Andrea; Villani, Daniela; Cipresso, Pietro; Repetto, Claudia; Serino, Silvia; Triberti, Stefano; Brivio, Eleonora; Galimberti, Carlo; Graffigna, Guendalina

    2014-01-01

    Information and communication technologies are widely and rapidly spreading in people's daily lives. But what is the possible role of the mass proliferation of digital devices in supporting healthy living and active ageing? Are they useful in fostering personal growth and individual integration of the elderly, by promoting satisfaction, opportunities for action, and self-expression? Rather, do they enhance automation, impose constraints on personal initiative, and result in compulsive consumption of information? In this chapter, we suggest that possible answers to these questions will be offered by the "Positive Technology" approach, i.e., the scientific and applied approach to using technology so that it improves the quality of our personal experiences through its structuring, augmentation, and/or replacement. First, we suggest that it is possible to use technology to manipulate the quality of experience with the goal of increasing wellness and generating strengths and resilience in individuals, organizations, and society. Then, we classify positive technologies according to their effects on these three features of personal experience - Hedonic: technologies used to induce positive and pleasant experiences; Eudaimonic: technologies used to support individuals in reaching engaging and self-actualizing experiences; Social/Interpersonal: technologies used to support and improve the connectedness between individuals, groups, and organizations. Finally, we discuss the possible role of positive technologies for healthy living and active ageing by presenting different practical applications of this approach.

  20. Positive Active Material For Alkaline Electrolyte Storage Battert Nickel Electrodes

    DOEpatents

    Bernard, Patrick; Baudry, Michelle

    2000-12-05

    A method of manufacturing a positive active material for nickel electrodes of alkaline storage batteries which consists of particles of hydroxide containing mainly nickel and covered with a layer of a hydroxide phase based on nickel and yttrium is disclosed. The proportion of the hydroxide phase is in the range 0.15% to 3% by weight of yttrium expressed as yttrium hydroxide relative to the total weight of particles.

  1. The effect of active-site isoleucine to alanine mutation on the DHFR catalyzed hydride-transfer

    PubMed Central

    Stojković, Vanja; Perissinotti, Laura L.; Lee, Jeeyeon; Benkovic, Stephen J.; Kohen, Amnon

    2015-01-01

    Comparison of the nature of hydride transfer in wild-type and active site mutant (I14A) of dihydrofolate reductase suggests that the size of this side chain at position 14 modulates H-tunneling. PMID:20972508

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

    SciTech Connect

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

    2007-01-01

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

  3. 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.

  4. 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.

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

    PubMed

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

    2010-12-01

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

  6. Dynamic positioning system based on active disturbance rejection technology

    NASA Astrophysics Data System (ADS)

    Lei, Zhengling; Guo, Chen; Fan, Yunsheng

    2015-08-01

    A dynamically positioned vessel, by the International Maritime Organization (IMO) and the certifying class societies (DNV, ABS, LR, etc.), is defined as a vessel that maintains its position and heading (fixed location or pre-determined track) exclusively by means of active thrusters. The development of control technology promotes the upgrading of dynamic positioning (DP) systems. Today there are two different DP systems solutions available on the market: DP system based on PID regulator and that based on model-based control. Both systems have limited disturbance rejection capability due to their design principle. In this paper, a new DP system solution is proposed based on Active Disturbance Rejection Control (ADRC) technology. This technology is composed of Tracking-Differentiator (TD), Extended State Observer (ESO) and Nonlinear Feedback Combination. On one hand, both TD and ESO can act as filters and can be used in place of conventional filters; on the other hand, the total disturbance of the system can be estimated and compensated by ESO, which therefore enhances the system's disturbance rejection capability. This technology's advantages over other methods lie in two aspects: 1) This method itself can not only achieve control objectives but also filter noisy measurements without other specialized filters; 2) This method offers a new useful approach to suppress the ocean disturbance. The simulation results demonstrate the effectiveness of the proposed method.

  7. Estimating snow water equivalent from GPS vertical site-position observations in the western United States

    PubMed Central

    Ouellette, Karli J; de Linage, Caroline; Famiglietti, James S

    2013-01-01

    [1] Accurate estimation of the characteristics of the winter snowpack is crucial for prediction of available water supply, flooding, and climate feedbacks. Remote sensing of snow has been most successful for quantifying the spatial extent of the snowpack, although satellite estimation of snow water equivalent (SWE), fractional snow covered area, and snow depth is improving. Here we show that GPS observations of vertical land surface loading reveal seasonal responses of the land surface to the total weight of snow, providing information about the stored SWE. We demonstrate that the seasonal signal in Scripps Orbit and Permanent Array Center (SOPAC) GPS vertical land surface position time series at six locations in the western United States is driven by elastic loading of the crust by the snowpack. GPS observations of land surface deformation are then used to predict the water load as a function of time at each location of interest and compared for validation to nearby Snowpack Telemetry observations of SWE. Estimates of soil moisture are included in the analysis and result in considerable improvement in the prediction of SWE. Citation: Ouellette, K. J., C. de Linage, and J. S. Famiglietti (2013), Estimating snow water equivalent from GPS vertical site-position observations in the western United States, Water Resour. Res., 49, 2508–2518, doi:10.1002/wrcr.20173. PMID:24223442

  8. Estimating snow water equivalent from GPS vertical site-position observations in the western United States.

    PubMed

    Ouellette, Karli J; de Linage, Caroline; Famiglietti, James S

    2013-05-01

    [1] Accurate estimation of the characteristics of the winter snowpack is crucial for prediction of available water supply, flooding, and climate feedbacks. Remote sensing of snow has been most successful for quantifying the spatial extent of the snowpack, although satellite estimation of snow water equivalent (SWE), fractional snow covered area, and snow depth is improving. Here we show that GPS observations of vertical land surface loading reveal seasonal responses of the land surface to the total weight of snow, providing information about the stored SWE. We demonstrate that the seasonal signal in Scripps Orbit and Permanent Array Center (SOPAC) GPS vertical land surface position time series at six locations in the western United States is driven by elastic loading of the crust by the snowpack. GPS observations of land surface deformation are then used to predict the water load as a function of time at each location of interest and compared for validation to nearby Snowpack Telemetry observations of SWE. Estimates of soil moisture are included in the analysis and result in considerable improvement in the prediction of SWE. Citation: Ouellette, K. J., C. de Linage, and J. S. Famiglietti (2013), Estimating snow water equivalent from GPS vertical site-position observations in the western United States, Water Resour. Res., 49, 2508-2518, doi:10.1002/wrcr.20173.

  9. BAX Activation is Initiated at a Novel Interaction Site

    PubMed Central

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

    2008-01-01

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

  10. Global Positioning System Synchronized Active Light Autonomous Docking System

    NASA Technical Reports Server (NTRS)

    Howard, Richard (Inventor)

    1994-01-01

    A Global Positioning System Synchronized Active Light Autonomous Docking System (GPSSALADS) for automatically docking a chase vehicle with a target vehicle comprises at least one active light emitting target which is operatively attached to the target vehicle. The target includes a three-dimensional array of concomitantly flashing lights which flash at a controlled common frequency. The GPSSALADS further comprises a visual tracking sensor operatively attached to the chase vehicle for detecting and tracking the target vehicle. Its performance is synchronized with the flash frequency of the lights by a synchronization means which is comprised of first and second internal clocks operatively connected to the active light target and visual tracking sensor, respectively, for providing timing control signals thereto, respectively. The synchronization means further includes first and second Global Positioning System receivers operatively connected to the first and second internal clocks, respectively, for repeatedly providing simultaneous synchronization pulses to the internal clocks, respectively. In addition, the GPSSALADS includes a docking process controller means which is operatively attached to the chase vehicle and is responsive to the visual tracking sensor for producing commands for the guidance and propulsion system of the chase vehicle.

  11. Global Positioning System Synchronized Active Light Autonomous Docking System

    NASA Technical Reports Server (NTRS)

    Howard, Richard T. (Inventor); Book, Michael L. (Inventor); Bryan, Thomas C. (Inventor); Bell, Joseph L. (Inventor)

    1996-01-01

    A Global Positioning System Synchronized Active Light Autonomous Docking System (GPSSALADS) for automatically docking a chase vehicle with a target vehicle comprising at least one active light emitting target which is operatively attached to the target vehicle. The target includes a three-dimensional array of concomitantly flashing lights which flash at a controlled common frequency. The GPSSALADS further comprises a visual tracking sensor operatively attached to the chase vehicle for detecting and tracking the target vehicle. Its performance is synchronized with the flash frequency of the lights by a synchronization means which is comprised of first and second internal clocks operatively connected to the active light target and visual tracking sensor, respectively, for providing timing control signals thereto, respectively. The synchronization means further includes first and second Global Positioning System receivers operatively connected to the first and second internal clocks, respectively, for repeatedly providing simultaneous synchronization pulses to the internal clocks, respectively. In addition, the GPSSALADS includes a docking process controller means which is operatively attached to the chase vehicle and is responsive to the visual tracking sensor for producing commands for the guidance and propulsion system of the chase vehicle.

  12. PLAP-1/Asporin Positively Regulates FGF-2 Activity.

    PubMed

    Awata, T; Yamada, S; Tsushima, K; Sakashita, H; Yamaba, S; Kajikawa, T; Yamashita, M; Takedachi, M; Yanagita, M; Kitamura, M; Murakami, S

    2015-10-01

    PLAP-1 is an extracellular matrix protein that is predominantly expressed in the periodontal ligament within periodontal tissue. It was previously revealed that PLAP-1 negatively regulates bone morphogenetic protein 2 and transforming growth factor β activity through direct interactions. However, the interaction between PLAP-1 and other growth factors has not been defined. Here, we revealed that PLAP-1 positively regulates the activity of fibroblast growth factor 2 (FGF-2), a critical growth factor in tissue homeostasis and repair. In this study, we isolated mouse embryonic fibroblasts (MEFs) from Plap-1(-/-) mice generated in our laboratory. Interestingly, Plap-1(-/-) MEFs exhibited enhanced responses to bone morphogenetic protein 2 but defective responses to FGF-2, and Plap-1 transfection into Plap-1(-/-) MEFs rescued these defective responses. In addition, binding assays revealed that PLAP-1 promotes FGF-2-FGF receptor 1 (FGFR1) complex formation by direct binding to FGF-2. Immunocytochemistry analyses revealed colocalization of PLAP-1 and FGF-2 in wild-type MEFs and reduced colocalization of FGF-2 and FGFR1 in Plap-1(-/-) MEFs compared with wild-type MEFs. Taken together, PLAP-1 positively regulates FGF-2 activity through a direct interaction. Extracellular matrix-growth factor interactions have considerable effects; thus, this approach may be useful in several regenerative medicine applications.

  13. Mitral valve repair for active culture positive infective endocarditis

    PubMed Central

    Doukas, G; Oc, M; Alexiou, C; Sosnowski, A W; Samani, N J; Spyt, T J

    2006-01-01

    Objective To describe the clinical and echocardiographic outcome after mitral valve (MV) repair for active culture positive infective MV endocarditis. Patients and methods Between 1996 and 2004, 36 patients (mean (SD) age 53 (18) years) with positive blood culture up to three weeks before surgery (or positive culture of material removed at operation) and intraoperative evidence of endocarditis underwent MV repair. Staphylococci and streptococci were the most common pathogens. All patients had moderate or severe mitral regurgitation (MR). Mean New York Heart Association (NYHA) class was 2.3 (1.0). Follow up was complete (mean 38 (19) months). Results Operative mortality was 2.8% (one patient). At follow up, endocarditis has not recurred. One patient developed severe recurrent MR and underwent valve replacement and one patient had moderate MR. There were two late deaths, both non‐cardiac. Kaplan‐Meier five year freedom from recurrent moderate to severe MR, freedom from repeat operation, and survival were 94 (4)%, 97 (3)%, and 93 (5)%, respectively. At the most recent review the mean NYHA class was 1.17 (0.3) (p < 0.0001). At the latest echocardiographic evaluation, left atrial diameters, left ventricular end diastolic diameter, and MV diameter were significantly reduced (p < 0.05) compared with preoperative values. Conclusions MV repair for active culture positive endocarditis is associated with low operative mortality and provides satisfactory freedom from recurrent infection, freedom from repeat operation, and survival. Hence, every effort should be made to repair infected MVs and valves should be replaced only when repair is not possible. PMID:15951395

  14. Involvement of novel autophosphorylation sites in ATM activation.

    PubMed

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

    2006-08-09

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

  15. 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.

  16. ON-LINE TOOLS FOR PROPER VERTICAL POSITIONING OF VERTICAL SAMPLING INTERVALS DURING SITE ASSESSMENT: MEETING IN MONTGOMERY, ALABAMA

    EPA Science Inventory

    This presentation, On-Line Tools for Proper Vertical Positioning of Sampling Intervals During Site Assessment, describes an approach to locating monitoring wells that is based on application of ground water models. The ideal use of both the model and site assessment funds is to ...

  17. 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…

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

    PubMed

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

    2014-12-16

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

  20. Roles of s3 site residues of nattokinase on its activity and substrate specificity.

    PubMed

    Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong

    2007-09-01

    Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent.

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

    PubMed

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

    2016-09-06

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

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

    PubMed Central

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

    2016-01-01

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

  3. Activation and repression functions of an SR splicing regulator depend on exonic versus intronic-binding position.

    PubMed

    Shen, Manli; Mattox, William

    2012-01-01

    SR proteins and related factors play widespread roles in alternative pre-mRNA splicing and are known to promote splice site recognition through their Arg-Ser-rich effector domains. However, binding of SR regulators to some targets results in repression of splice sites through a distinct mechanism. Here, we investigate how activated and repressed targets of the Drosophila SR regulator Transformer2 elicit its differing effects on splicing. We find that, like activation, repression affects early steps in the recognition of splice sites and spliceosome assembly. Repositioning of regulatory elements reveals that Tra2 complexes that normally repress splicing from intronic positions activate splicing when located in an exon. Protein tethering experiments demonstrate that this position dependence is an intrinsic property of Tra2 and further show that repression and activation are mediated by separate effector domains of this protein. When other Drosophila SR factors (SF2 and Rbp1) that activate splicing from exonic positions were tethered intronically they failed to either activate or repress splicing. Interestingly, both activities of Tra2 favor the exonic identity of the RNA sequences that encompass its binding sites. This suggests a model in which these two opposite functions act in concert to define both the position and extent of alternatively spliced exons.

  4. Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

    SciTech Connect

    Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.; Elsen, Nathaniel L.; Phillips, Jr., George N.; Fox, Brian G.

    2014-10-02

    Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric {mu}-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH{sub 2}-benzoate and p-Br-benzoate showed a {mu}-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a {pi}-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 {angstrom}) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential

  5. Prevalence of avian haemosporidian parasites is positively related to the abundance of host species at multiple sites within a region.

    PubMed

    Ellis, Vincenzo A; Medeiros, Matthew C I; Collins, Michael D; Sari, Eloisa H R; Coffey, Elyse D; Dickerson, Rebecca C; Lugarini, Camile; Stratford, Jeffrey A; Henry, Donata R; Merrill, Loren; Matthews, Alix E; Hanson, Alison A; Roberts, Jackson R; Joyce, Michael; Kunkel, Melanie R; Ricklefs, Robert E

    2017-01-01

    Parasite prevalence is thought to be positively related to host population density owing to enhanced contagion. However, the relationship between prevalence and local abundance of multiple host species is underexplored. We surveyed birds and their haemosporidian parasites (genera Plasmodium and Haemoproteus) at multiple sites across eastern North America to test whether the prevalence of these parasites in a host species at a particular site is related to that host's local abundance. Prevalence was positively related to host abundance within most sites, although the effect was stronger and more consistent for Plasmodium than for Haemoproteus. In contrast, prevalence was not related to variation in the abundance of most individual host species among sites across the region. These results suggest that parasite prevalence partly reflects the relative abundances of host species in local assemblages. However, three nonnative host species had low prevalence despite being relatively abundant at one site, as predicted by the enemy release hypothesis.

  6. Sources identification of the atmospheric aerosol at urban and suburban sites in Indonesia by positive matrix factorization.

    PubMed

    Santoso, Muhayatun; Hopke, Philip K; Hidayat, Achmad; Diah Dwiana L

    2008-07-01

    Samples of fine and coarse fractions of airborne particulate matter were collected in Indonesia (west central Java) at an urban site in Bandung and in suburban Lembang from January 2002 to December 2004. The samples were collected using a Gent stacked filter sampler in two size fractions of <2.5 microm (fine) and 2.5 to 10 microm (coarse). The samples were analyzed for elemental concentrations by instrumental neutron activation analysis (INAA) and proton-induced X-ray emission (PIXE). Black carbon was determined using an EEL Smoke Stain Reflectometer. The data sets were then analyzed using positive matrix factorization to identify the possible sources of fine and coarse atmospheric aerosols in both areas. The best solutions were found to be seven factors and five factors for elemental compositions of fine and coarse particulate matter in the urban area of Bandung and six factors and five factors for elemental compositions of fine and coarse particulate matter in the suburban area of Lembang, respectively. The sources are soil dust, motor vehicles, biomass burning, sea salt, and road dust. The PMF results showed that more than 50% of the PM2.5-10 mass at both sites comes from soil dust and road dust. The biomass burning factor contributes about 40% of the PM2.5 mass in case of suburban Lembang and about 20% in urban Bandung.

  7. Requirement of helix P2.2 and nucleotide G1 for positioning the cleavage site and cofactor of the glmS ribozyme.

    PubMed

    Klein, Daniel J; Wilkinson, Sara R; Been, Michael D; Ferré-D'Amaré, Adrian R

    2007-10-12

    The glmS ribozyme is a catalytic RNA that self-cleaves at its 5'-end in the presence of glucosamine 6-phosphate (GlcN6P). We present structures of the glmS ribozyme from Thermoanaerobacter tengcongensis that are bound with the cofactor GlcN6P or the inhibitor glucose 6-phosphate (Glc6P) at 1.7 A and 2.2 A resolution, respectively. The two structures are indistinguishable in the conformations of the small molecules and of the RNA. GlcN6P binding becomes apparent crystallographically when the pH is raised to 8.5, where the ribozyme conformation is identical with that observed previously at pH 5.5. A key structural feature of this ribozyme is a short duplex (P2.2) that is formed between sequences just 3' of the cleavage site and within the core domain, and which introduces a pseudoknot into the active site. Mutagenesis indicates that P2.2 is required for activity in cis-acting and trans-acting forms of the ribozyme. P2.2 formation in a trans-acting ribozyme was exploited to demonstrate that N1 of the guanine at position 1 contributes to GlcN6P binding by interacting with the phosphate of the cofactor. At neutral pH, RNAs with adenine, 2-aminopurine, dimethyladenine or purine substitutions at position 1 cleave faster with glucosamine than with GlcN6P. This altered cofactor preference provides biochemical support for the orientation of the cofactor within the active site. Our results establish two features of the glmS ribozyme that are important for its activity: a sequence within the core domain that selects and positions the cleavage-site sequence, and a nucleobase at position 1 that helps position GlcN6P.

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

    PubMed

    Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

    2016-10-01

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

  9. Rapid deamination of cyclobutane pyrimidine dimer photoproducts at TCG sites in a translationally and rotationally positioned nucleosome in vivo.

    PubMed

    Cannistraro, Vincent J; Pondugula, Santhi; Song, Qian; Taylor, John-Stephen

    2015-10-30

    Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T(m)CG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots.

  10. Face the Edges: Catalytic Active Sites of Nanomaterials

    PubMed Central

    Ni, Bing

    2015-01-01

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

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

    PubMed

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

    2015-04-21

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

  12. 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.

  13. 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.

  14. 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

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

    PubMed

    Davis, A E

    1980-08-01

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

  16. 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.

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

    PubMed

    Li, Lifeng; Ni, Ye; Sun, Zhihao

    2012-04-01

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

  18. Do release-site biases reflect response to the Earth's magnetic field during position determination by homing pigeons?

    PubMed

    Mora, Cordula V; Walker, Michael M

    2009-09-22

    How homing pigeons (Columba livia) return to their loft from distant, unfamiliar sites has long been a mystery. At many release sites, untreated birds consistently vanish from view in a direction different from the home direction, a phenomenon called the release-site bias. These deviations in flight direction have been implicated in the position determination (or map) step of navigation because they may reflect local distortions in information about location that the birds obtain from the geophysical environment at the release site. Here, we performed a post hoc analysis of the relationship between vanishing bearings and local variations in magnetic intensity using previously published datasets for pigeons homing to lofts in Germany. Vanishing bearings of both experienced and naïve birds were strongly associated with magnetic intensity variations at release sites, with 90 per cent of bearings lying within +/-29 degrees of the magnetic intensity slope or contour direction. Our results (i) demonstrate that pigeons respond in an orderly manner to the local structure of the magnetic field at release sites, (ii) provide a mechanism for the occurrence of release-site biases and (iii) suggest that pigeons may derive spatial information from the magnetic field at the release site that could be used to estimate their current position relative to their loft.

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

    PubMed

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

    2002-01-31

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

  20. Arylesterase Phenotype-Specific Positive Association Between Arylesterase Activity and Cholinesterase Specific Activity in Human Serum

    PubMed Central

    Aoki, Yutaka; Helzlsouer, Kathy J.; Strickland, Paul T.

    2014-01-01

    Context: Cholinesterase (ChE) specific activity is the ratio of ChE activity to ChE mass and, as a biomarker of exposure to cholinesterase inhibitors, has a potential advantage over simple ChE activity. Objective: To examine the association of several potential correlates (serum arylesterase/paraoxonase activity, serum albumin, sex, age, month of blood collection, and smoking) with plasma ChE specific activity. Methods: We analyzed data from 195 cancer-free controls from a nested case-control study, accounting for potential confounding. Results: Arylesterase activity had an independent, statistically significant positive association with ChE specific activity, and its magnitude was the greatest for the arylesterase phenotype corresponding to the QQ PON1192 genotype followed by phenotypes corresponding to QR and RR genotypes. Serum albumin was positively associated with ChE specific activity. Conclusions: Plasma arylesterase activity was positively associated with plasma ChE specific activity. This observation is consistent with protection conferred by a metabolic phenotype resulting in reduced internal dose. PMID:24473115

  1. MSK1 activity is controlled by multiple phosphorylation sites

    PubMed Central

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

    2004-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1991-11-01

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

  3. Identification of highly reactive cysteine residues at less exposed positions in the Fab constant region for site-specific conjugation.

    PubMed

    Shiraishi, Yasuhisa; Muramoto, Takashige; Nagatomo, Kazutaka; Shinmi, Daisuke; Honma, Emiko; Masuda, Kazuhiro; Yamasaki, Motoo

    2015-06-17

    Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.

  4. 'The positive feel': Unpacking the role of positive thinking in people with multiple sclerosis's thinking aloud about staying physically active.

    PubMed

    Hall-McMaster, Samuel M; Treharne, Gareth J; Smith, Catherine M

    2016-12-01

    People with multiple sclerosis experience barriers to physical activity. Thought processes are interwoven with garnering motivation to overcome these barriers. This study investigated in-depth the role of positive thinking in physical activity motivation of two women and two men with multiple sclerosis. Participants thought aloud while completing standardised measures of physical activity, stages of change and self-efficacy, and in response to planned and spontaneous questions. Four themes were formulated using inductive thematic analysis: thoughts about purpose, self-efficacy, the past and reinforcement through positive thinking. These findings have implications for physical activity theories and delivering appropriate physical activity interventions to the multiple sclerosis community.

  5. 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.

  6. 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

  7. Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation

    PubMed Central

    Zhang, Hua; Song, Lei; Cong, Haolong

    2015-01-01

    ABSTRACT Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5′ untranslated region (5′UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. IMPORTANCE The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection

  8. 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.

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

    PubMed

    Goodstadt, Leo; Ponting, Chris P

    2004-06-01

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

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

    PubMed

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

    2007-07-21

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

  11. Infiltration of PD-1-positive cells in combination with tumor site PD-L1 expression is a positive prognostic factor in cutaneous angiosarcoma.

    PubMed

    Honda, Yuki; Otsuka, Atsushi; Ono, Sachiko; Yamamoto, Yosuke; Seidel, Judith A; Morita, Satoshi; Hirata, Masahiro; Kataoka, Tatsuki R; Takenouchi, Tatsuya; Fujii, Kazuyasu; Kanekura, Takuro; Okubo, Yuko; Takahashi, Kenzo; Yanagi, Teruki; Hoshina, Daichi; Hata, Hiroo; Abe, Riichiro; Fujimura, Taku; Funakoshi, Takeru; Yoshino, Koji; Masuzawa, Mamiko; Amoh, Yasuyuki; Tanaka, Ryota; Fujisawa, Yasuhiro; Honda, Tetsuya; Kabashima, Kenji

    2017-01-01

    Cutaneous angiosarcoma (CAS) is a malignant sarcoma with poor prognosis. Programmed cell death-1 (PD-1)/programmed cell death-1 ligand-1 (PD-L1) expression reflects antitumor immunity, and is associated with patient prognosis in various cancers. The purpose of this study is to investigate the relationship between PD-1/PD-L1 expression and CAS prognosis. CAS cases (n = 106) were immunohistochemically studied for PD-L1 and PD-1 expression, and the correlation with patient prognosis was analyzed. PD-L1 expression was assessed by flow cytometry on three CAS cell lines with or without IFNγ stimulation. A total of 30.2% of patients' samples were positive for PD-L1, and 17.9% showed a high infiltration of PD-1-positive cells. Univariate analysis showed a significant relationship between a high infiltration of PD-1-positive cells with tumor site PD-L1 expression and favorable survival in stage 1 patients (p = 0.014, log-rank test). Multivariable Cox-proportional hazard regression analysis also showed that patients with a high infiltration of PD-1-positive cells with tumor site PD-L1 expression were more likely to have favorable survival, after adjustment with possible confounders (hazard ratio (HR) = 0.38, p = 0.021, 95% confidence interval (CI) 0.16-0.86). Immunofluorescence staining of CAS samples revealed that PD-L1-positive cells were adjacent to PD-1-positive cells and/or tumor stroma with high IFNγ expression. In vitro stimulation with IFNγ increased PD-L1 expression in two out of three established CAS cell lines. Our results suggest that PD-1/PD-L1 expression is related to CAS progression, and the treatment with anti-PD-1 antibodies could be a new therapeutic option for CAS.

  12. 5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB).

    PubMed Central

    Giono, L E; Varone, C L; Cánepa, E T

    2001-01-01

    The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression. PMID:11139395

  13. 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

  14. 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

  15. Molecular mimicry of substrate oxygen atoms by water molecules in the beta-amylase active site.

    PubMed

    Pujadas, G; Palau, J

    2001-08-01

    Soybean beta-amylase (EC 3.2.1.2) has been crystallized both free and complexed with a variety of ligands. Four water molecules in the free-enzyme catalytic cleft form a multihydrogen-bond network with eight strategic residues involved in enzyme-ligand hydrogen bonds. We show here that the positions of these four water molecules are coincident with the positions of four potential oxygen atoms of the ligands within the complex. Some of these waters are displaced from the active site when the ligands bind to the enzyme. How many are displaced depends on the shape of the ligand. This means that when one of the four positions is not occupied by a ligand oxygen atom, the corresponding water remains. We studied the functional/structural role of these four waters and conclude that their presence means that the conformation of the eight side chains is fixed in all situations (free or complexed enzyme) and preserved from unwanted or forbidden conformational changes that could hamper the catalytic mechanism. The water structure at the active pocket of beta-amylase is therefore essential for providing the ligand recognition process with plasticity. It does not affect the protein active-site geometry and preserves the overall hydrogen-bonding network, irrespective of which ligand is bound to the enzyme. We also investigated whether other enzymes showed a similar role for water. Finally, we discuss the potential use of these results for predicting whether water molecules can mimic ligand atoms in the active center.

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed Central

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

    2016-01-01

    Fructose 1,6-bisphosphate aldolase (FBA) is important for both glycolysis and gluconeogenesis in life. Class II (zinc dependent) FBA is an attractive target for the development of antibiotics against protozoa, bacteria, and fungi, and is also widely used to produce various high-value stereoisomers in the chemical and pharmaceutical industry. In this study, the crystal structures of class II Escherichia coli FBA (EcFBA) were determined from four different crystals, with resolutions between 1.8 Å and 2.0 Å. Native EcFBA structures showed two separate sites of Zn1 (interior position) and Zn2 (active site surface position) for Zn2+ ion. Citrate and TRIS bound EcFBA structures showed Zn2+ position exclusively at Zn2. Crystallographic snapshots of EcFBA structures with and without ligand binding proposed the rationale of metal shift at the active site, which might be a hidden mechanism to keep the trace metal cofactor Zn2+ within EcFBA without losing it. PMID:27733232

  18. Absolute Side-chain Structure at Position 13 Is Required for the Inhibitory Activity of Bromein*

    PubMed Central

    Sawano, Yoriko; Hatano, Ken-ichi; Miyakawa, Takuya; Tanokura, Masaru

    2008-01-01

    Bromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu13 in the light chain was revealed to be essential for inhibition. Pro12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed φ dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu13 are absolutely crucial for bromelain inhibition. PMID:18948264

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

    PubMed Central

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

    2009-01-01

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

  20. 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.

  1. Position paper - peer review and design verification of selected activities

    SciTech Connect

    Stine, M.D.

    1994-09-01

    Position Paper to develop and document a position on the performance of independent peer reviews on selected design and analysis components of the Title I (preliminary) and Title II (detailed) design phases of the Multi-Function Waste Tank Facility project.

  2. Small Molecule Positive Allosteric Modulation of TRPV1 Activation by Vanilloids and Acidic pHS⃞

    PubMed Central

    Kaszas, Krisztian; Keller, Jason M.; Coddou, Claudio; Mishra, Santosh K.; Hoon, Mark A.; Stojilkovic, Stanko; Jacobson, Kenneth A.

    2012-01-01

    Transient receptor potential cation channel subfamily V member 1 (TRPV1) is a high-conductance, nonselective cation channel strongly expressed in nociceptive primary afferent neurons of the peripheral nervous system and functions as a multimodal nociceptor gated by temperatures greater than 43°C, protons, and small-molecule vanilloid ligands such as capsaicin. The ability to respond to heat, low pH, vanilloids, and endovanilloids and altered sensitivity and expression in experimental inflammatory and neuropathic pain models made TRPV1 a major target for the development of novel, nonopioid analgesics and resulted in the discovery of potent antagonists. In human clinical trials, observations of hyperthermia and the potential for thermal damage by suppressing the ability to sense noxious heat suggested that full-scale blockade of TRPV1 function can be counterproductive and subtler pharmacological approaches are necessary. Here we show that the dihydropyridine derivative 4,5-diethyl-3-(2-methoxyethylthio)-2-methyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1477) behaves as a positive allosteric modulator of both proton and vanilloid activation of TRPV1. Under inflammatory-mimetic conditions of low pH (6.0) and protein kinase C phosphorylation, addition of MRS1477 further increased sensitivity of already sensitized TPRV1 toward capsaicin. MRS1477 does not affect inhibition by capsazepine or ruthenium red and remains effective in potentiating activation by pH in the presence of an orthosteric vanilloid antagonist. These results indicate a distinct site on TRPV1 for positive allosteric modulation that may bind endogenous compounds or novel pharmacological agents. Positive modulation of TRPV1 sensitivity suggests that it may be possible to produce a selective analgesia through calcium overload restricted to highly active nociceptive nerve endings at sites of tissue damage and inflammation. PMID:22005042

  3. Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model

    PubMed Central

    Huang, Xiaoqiang; Xue, Jing; Lin, Min; Zhu, Yushan

    2016-01-01

    Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic

  4. Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site*

    PubMed Central

    Dahal, Rejwi Acharya; Pramod, Akula Bala; Sharma, Babita; Krout, Danielle; Foster, James D.; Cha, Joo Hwan; Cao, Jianjing; Newman, Amy Hauck; Lever, John R.; Vaughan, Roxanne A.; Henry, L. Keith

    2014-01-01

    The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4′-azido-3′-iodophenylethyl ester ([125I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [125I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors. PMID:25179220

  5. Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site.

    PubMed

    Dahal, Rejwi Acharya; Pramod, Akula Bala; Sharma, Babita; Krout, Danielle; Foster, James D; Cha, Joo Hwan; Cao, Jianjing; Newman, Amy Hauck; Lever, John R; Vaughan, Roxanne A; Henry, L Keith

    2014-10-24

    The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([(125)I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [(125)I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.

  6. Active-site modifications of adenylation domains lead to hydrolysis of upstream nonribosomal peptidyl thioester intermediates.

    PubMed

    Uguru, Gabriel C; Milne, Claire; Borg, Matthew; Flett, Fiona; Smith, Colin P; Micklefield, Jason

    2004-04-28

    Site-directed mutagenesis of nonribosomal peptide synthetase (NRPS) adenylation (A) domains was investigated as a means to engineer new calcium-dependent antibiotics (CDA) in Streptomyces coelicolor. Single- and double-point mutants of the CDA NRPS module 7, A-domain were generated, which were predicted to alter the specificity of this domain from Asp to Asn. The double-point mutant produced a new peptide CDA2a-7N containing Asn at position 7 as expected. However, in both the single- and the double-point mutants, significant hydrolysis of the CDA-6mer intermediate was evident. One explanation for this is that the mutant module 7 A-domain activates Asn instead of Asp; however, the Asn-thioester intermediate is only weakly recognized by the upstream C-domain acceptor site (a), allowing a water molecule to intercept the hexapeptidyl intermediate in the donor site (d).

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

    PubMed

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

    2017-03-01

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

  8. 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.

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

    PubMed

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

    2011-07-10

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

  10. Active Healthy Kids Canada's Position on Active Video Games for Children and Youth.

    PubMed

    Chaput, Jean-Philippe; Leblanc, Allana G; McFarlane, Allison; Colley, Rachel C; Thivel, David; Biddle, Stuart Jh; Maddison, Ralph; Leatherdale, Scott T; Tremblay, Mark S

    2013-12-01

    The effect of active video games (AVGs) on acute energy expenditure has previously been reported; however, the influence of AVGs on other health-related lifestyle indicators remains unclear. To address this knowledge gap, Active Healthy Kids Canada (AHKC) convened an international group of researchers to conduct a systematic review to understand whether AVGs should be promoted to increase physical activity and improve health indicators in children and youth (zero to 17 years of age). The present article outlines the process and outcomes of the development of the AHKC's position on active video games for children and youth. In light of the available evidence, AHKC does not recommend AVGs as a strategy to help children be more physically active. However, AVGs may exchange some sedentary time for light- to moderate-intensity physical activity, and there may be specific situations in which AVGs provide benefit (eg, motor skill development in special populations and rehabilitation).

  11. Cdc42 activation couples spindle positioning to first polar body formation in oocyte maturation.

    PubMed

    Ma, Chunqi; Benink, Héléne A; Cheng, Daye; Montplaisir, Véronique; Wang, Ling; Xi, Yanwei; Zheng, Pei-Pei; Bement, William M; Liu, X Johné

    2006-01-24

    During vertebrate egg maturation, cytokinesis initiates after one pole of the bipolar metaphase I spindle attaches to the oocyte cortex, resulting in the formation of a polar body and the mature egg. It is not known what signal couples the spindle pole positioning to polar body formation. We approached this question by drawing an analogy to mitotic exit in budding yeast, as asymmetric spindle attachment to the appropriate cortical region is the common regulatory cue. In budding yeast, the small G protein Cdc42 plays an important role in mitotic exit following the spindle pole attachment . We show here that inhibition of Cdc42 activation blocks polar body formation. The oocytes initiate anaphase but fail to properly form and direct a contractile ring. Endogenous Cdc42 is activated at the spindle pole-cortical contact site immediately prior to polar body formation. The cortical Cdc42 activity zone, which directly overlays the spindle pole, is circumscribed by a cortical RhoA activity zone; the latter defines the cytokinetic contractile furrow . As the RhoA ring contracts during cytokinesis, the Cdc42 zone expands, maintaining its complementary relationship with the RhoA ring. Cdc42 signaling may thus be an evolutionarily conserved mechanism that couples spindle positioning to asymmetric cytokinesis.

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

    PubMed

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

    2016-11-16

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

  13. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

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

    SciTech Connect

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

    1992-02-01

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

  16. Determination of the positions of aluminum atoms introduced into SSZ-35 and the catalytic properties of the generated Brønsted acid sites.

    PubMed

    Miyaji, Akimitsu; Kimura, Nobuhiro; Shiga, Akinobu; Hayashi, Yoshihiro; Nishitoba, Toshiki; Motokura, Ken; Baba, Toshihide

    2017-03-01

    The positions of aluminum (Al) atoms in SSZ-35 together with the characteristics of the generated protons were investigated by (27)Al multiple quantum magic-angle spinning (MQ-MAS), (29)Si MAS, and (1)H MAS NMR data analyses accompanied by a variable temperature (1)H MAS NMR analysis. The origin of the acidic -OH groups (Brønsted acid sites) generated by introducing Al atoms into the T sites was investigated and the T sites introduced into the Al atoms were revealed. To further determine the catalytic properties of the acidic protons generated in SSZ-35, the influence of the concentration of the Al atoms on the catalytic activity and selectivity during the transformation of toluene was examined.

  17. Promoting Teachers' Positive Attitude towards Web Use: A Study in Web Site Development

    ERIC Educational Resources Information Center

    Akpinar, Yavuz; Bayramoglu, Yusuf

    2008-01-01

    The purpose of the study was to examine effects of a compact training for developing web sites on teachers' web attitude, as composed of: web self efficacy, perceived web enjoyment, perceived web usefulness and behavioral intention to use the web. To measure the related constructs, the Web Attitude Scale was adapted into Turkish and tested with a…

  18. Position-specific prediction of methylation sites from sequence conservation based on information theory.

    PubMed

    Shi, Yinan; Guo, Yanzhi; Hu, Yayun; Li, Menglong

    2015-07-23

    Protein methylation plays vital roles in many biological processes and has been implicated in various human diseases. To fully understand the mechanisms underlying methylation for use in drug design and work in methylation-related diseases, an initial but crucial step is to identify methylation sites. The use of high-throughput bioinformatics methods has become imperative to predict methylation sites. In this study, we developed a novel method that is based only on sequence conservation to predict protein methylation sites. Conservation difference profiles between methylated and non-methylated peptides were constructed by the information entropy (IE) in a wider neighbor interval around the methylation sites that fully incorporated all of the environmental information. Then, the distinctive neighbor residues were identified by the importance scores of information gain (IG). The most representative model was constructed by support vector machine (SVM) for Arginine and Lysine methylation, respectively. This model yielded a promising result on both the benchmark dataset and independent test set. The model was used to screen the entire human proteome, and many unknown substrates were identified. These results indicate that our method can serve as a useful supplement to elucidate the mechanism of protein methylation and facilitate hypothesis-driven experimental design and validation.

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

    PubMed Central

    Rothmel, R K; LeClerc, J E

    1989-01-01

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

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

    PubMed Central

    Ju, Kou-San; Parales, Rebecca E.

    2006-01-01

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

  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. 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.

  3. 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.

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

    PubMed

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

    2010-07-14

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

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

    PubMed

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

    2014-09-01

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

  6. Design and synthesis of new dihydrotestosterone derivative with positive inotropic activity.

    PubMed

    Lauro, Figueroa-Valverde; Francisco, Díaz-Cedillo; Elodia, García-Cervera; Eduardo, Pool-Gómez; Marcela, Rosas-Nexticapa; Lenin, Hau-Heredia; Betty, Sarabia Alcocer

    2015-03-01

    There are several reports which indicate that some steroid derivatives have inotropic activity; nevertheless, the cellular site and mechanism of action of steroid derivatives at cardiovascular level is very confusing. In order, to clarify these phenomena in this study, two dihydrotestosterone derivatives (compounds 5 and 10) were synthesized with the objective of to evaluate its biological activity on left ventricular pressure and characterize their molecular mechanism. In the first stage, the Langendorff technique was used to measure changes on perfusion pressure and coronary resistance in an isolated rat heart model in absence or presence of the steroid derivatives. Additionally, to characterize the molecular mechanism involved in the inotropic activity induced by the compound 5 was evaluated by measuring left ventricular pressure in absence or presence of following compounds; nifedipine, flutamide, indomethacin, prazosin, isoproterenol, propranolol and metoprolol. The results showed that the compound 5 significantly increased the perfusion pressure and coronary resistance in comparison with dihydrotestosterone, compound 10 and the control conditions. Other data indicate that 5 increase left ventricular pressure in a dose-dependent manner (0.001-100 nM); nevertheless, this phenomenon was significantly inhibited only by propranolol or metoprolol at a dose of 1 nM. These data suggest that positive inotropic activity induced by the compound 5 is through β1-adrenergic receptor however, this effect was independent of cAMP levels. This phenomenon is a particularly interesting because the positive inotropic activity induced by this steroid derivative involves a molecular mechanism different in comparison with other positive inotropic drugs.

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

    PubMed Central

    Arthur, J S; Elce, J S

    1996-01-01

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

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

    PubMed

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

    2013-02-01

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

  9. Different cleavage sites are aligned differently in the active site of M1 RNA, the catalytic subunit of Escherichia coli RNase P.

    PubMed Central

    Kufel, J; Kirsebom, L A

    1996-01-01

    We have studied RNase P RNA (M1 RNA) cleavage of model tRNA precursors that are cleaved at two independent positions. Here we present data demonstrating that cleavage at both sites depends on the 2'-OH immediately 5' of the respective cleavage site. However, we show that the 2-amino group of a guanosine at the cleavage site plays a significant role in cleavage at one of these sites but not at the other. These data suggest that these two cleavage sites are handled differently by the ribozyme. This theory is supported by our finding that the cross-linking pattern between Ml RNA and tRNA precursors carrying 4-thioU showed distinct differences, depending on the location of the 4-thioU relative to the respective cleavage site. These findings lead us to suggest that different cleavage sites are aligned differently in the active site, possibly as a result of different binding modes of a substrate to M1 RNA. We discuss a model in which the interaction between the 3'-terminal "RCCA" motif (first three residues interact) of a tRNA precursor and M1 RNA plays a significant role in this process. Images Fig. 2 Fig. 3 Fig. 4 PMID:8650223

  10. Collecting "Total" Vocational Education and Training Activity. Position Paper

    ERIC Educational Resources Information Center

    Karmel, Tom

    2011-01-01

    In this position paper, NCVER's Managing Director, Dr Tom Karmel, argues that the submission of vocational education and training student data should be mandated as a condition of registration for all registered training organisations, including private providers. This will ensure a comprehensive data collection that gives a realistic view of…

  11. Positive Youth Development through Physical Activity: Opportunities for Physical Educators

    ERIC Educational Resources Information Center

    Hemphill, Michael A.

    2014-01-01

    As physical educators continue to advocate for school-based PE, they should also consider ways to extend their work into community settings in an effort to ensure that all kids have an opportunity to develop physical literacy. This article describes how positive youth development programs can provide an opportunity for physical educators to engage…

  12. Positive feedback of protein kinase C proteolytic activation during apoptosis.

    PubMed Central

    Leverrier, Sabrina; Vallentin, Alice; Joubert, Dominique

    2002-01-01

    In contrast with protein kinase Calpha (PKCalpha) and PKCepsilon, which are better known for promoting cell survival, PKCdelta is known for its pro-apoptotic function, which is exerted mainly through a caspase-3-dependent proteolytic activation pathway. In the present study, we used the rat GH3B6 pituitary adenoma cell line to show that PKCalpha and PKCepsilon are activated and relocalized together with PKCdelta when apoptosis is induced by a genotoxic stress. Proteolytic activation is a crucial step used by the three isoforms since: (1) the catalytic domains of the PKCalpha, PKCepsilon or PKCdelta isoforms (CDalpha, CDepsilon and CDdelta respectively) accumulated, and this accumulation was dependent on the activity of both calpain and caspase; and (2) transient expression of CDalpha, CDepsilon or CDdelta sufficed to induce apoptosis. However, following this initial step of proteolytic activation, the pathways diverge; cytochrome c release and caspase-3 activation are induced by CDepsilon and CDdelta, but not by CDalpha. Another interesting finding of the present study is the proteolysis of PKCdelta induced by CDepsilon expression that revealed the existence of a cross-talk between PKC isoforms during apoptosis. Hence the PKC family may participate in the apoptotic process of pituitary adenoma cells at two levels: downstream of caspase and calpain, and via retro-activation of caspase-3, resulting in the amplification of its own proteolytic activation. PMID:12238950

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

    SciTech Connect

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

    2011-08-16

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

  14. The position of mitochondria and ER in relation to that of the secretory sites in chromaffin cells.

    PubMed

    Villanueva, José; Viniegra, Salvador; Gimenez-Molina, Yolanda; García-Martinez, Virginia; Expósito-Romero, Giovanna; del Mar Frances, Maria; García-Sancho, Javier; Gutiérrez, Luis M

    2014-12-01

    Knowledge of the distribution of mitochondria and endoplasmic reticulum (ER) in relation to the position of exocytotic sites is relevant to understanding the influence of these organelles in tuning Ca(2+) signals and secretion. Confocal images of probes tagged to mitochondria and the F-actin cytoskeleton revealed the existence of two populations of mitochondria, one that was cortical and one that was perinuclear. This mitochondrial distribution was also confirmed by using electron microscopy. In contrast, ER was sparse in the cortex and more abundant in deep cytoplasmic regions. The mitochondrial distribution might be due to organellar transport, which experiences increasing restrictions in the cell cortex. Further study of organelle distribution in relation to the position of SNARE microdomains and the granule fusion sites revealed that a third of the cortical mitochondria colocalized with exocytotic sites and another third located at a distance closer than two vesicle diameters. ER structures were also present in the vicinity of secretory sites but at a lower density. Therefore, mitochondria and ER have a spatial distribution that suggests a specialized role in modulation of exocytosis that fits with the role of cytosolic Ca(2+) microdomains described previously.

  15. Positive inotropic activity induced by a dehydroisoandrosterone derivative in isolated rat heart model.

    PubMed

    Figueroa-Valverde, L; Díaz-Cedillo, F; García-Cervera, E; Pool Gómez, E; López-Ramos, M; Rosas-Nexticapa, M; Martinez-Camacho, R

    2013-10-01

    Experimental studies indicate that some steroid derivatives have inotropic activity; nevertheless, there is scarce information about the effects of the dehydroisoandrosterone and its derivatives at cardiovascular level. In addition, to date the cellular site and mechanism of action of dehydroisoandrosterone at cardiovascular level is very confusing. In order, to clarify those phenomena in this study, a dehydroisoandrosterone derivative was synthesized with the objective of to evaluate its activity on perfusion pressure and coronary resistance and compare this phenomenon with the effect exerted by dehydroisoandrosterone. The Langendorff technique was used to measure changes on perfusion pressure and coronary resistance in an isolated rat heart model in absence or presence of dehydroisoandrosterone and its derivative. Additionally, to characterize the molecular mechanism involved in the inotropic activity induced by dehydroisoandrosterone derivative was evaluated by measuring left ventricular pressure in absence or presence of following compounds; flutamide, prazosin, metoprolol and nifedipine. The results showed that dehydroisoandrosterone derivative significantly increased the perfusion pressure and coronary resistance in comparison with the control conditions and dehydroisoandrosterone. Additionally, other data indicate that dehydroisoandrosterone derivative increase left ventricular pressure in a dose-dependent manner [1 × 10(-9)-1 × 10(-4) mmol]; nevertheless, this phenomenon was significantly inhibited by nifedipine at a dose of 1 × 10(-6) mmol. In conclusion, these data suggest that dehydroisoandrosterone derivative induces positive inotropic activity through of activation the L-type calcium channel.

  16. Molecular cloning, characterization and positively selected sites of the glutathione S-transferase family from Locusta migratoria.

    PubMed

    Zhang, Xueyao; Wang, Jianxin; Zhang, Min; Qin, Guohua; Li, Daqi; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2014-01-01

    Glutathione S-transferases (GSTs) are multifunctional enzymes that are involved in the metabolism of endogenous and exogenous compounds and are related to insecticide resistance. The purpose of this study was to provide new information on the molecular characteristics and the positive selection of locust GSTs. Based on the transcriptome database, we sequenced 28 cytosolic GSTs and 4 microsomal GSTs from the migratory locust (Locusta migratoria). We assigned the 28 cytosolic GSTs into 6 classes--sigma, epsilon, delta, theta, omega and zeta, and the 4 microsomal GSTs into 2 subclasses--insect and MGST3. The tissue- and stage-expression patterns of the GSTs differed at the mRNA level. Further, the substrate specificities and kinetic constants of the cytosolic GSTs differed markedly at the protein level. The results of likelihood ratio tests provided strong evidence for positive selection in the delta class. The result of Bayes Empirical Bayes analysis identified 4 amino acid sites in the delta class as positive selection sites. These sites were located on the protein surface. Our findings will facilitate the elucidation of the molecular characteristics and evolutionary aspects of insect GST superfamily.

  17. Molecular Cloning, Characterization and Positively Selected Sites of the Glutathione S-Transferase Family from Locusta migratoria

    PubMed Central

    Zhang, Min; Qin, Guohua; Li, Daqi; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2014-01-01

    Glutathione S-transferases (GSTs) are multifunctional enzymes that are involved in the metabolism of endogenous and exogenous compounds and are related to insecticide resistance. The purpose of this study was to provide new information on the molecular characteristics and the positive selection of locust GSTs. Based on the transcriptome database, we sequenced 28 cytosolic GSTs and 4 microsomal GSTs from the migratory locust (Locusta migratoria). We assigned the 28 cytosolic GSTs into 6 classes—sigma, epsilon, delta, theta, omega and zeta, and the 4 microsomal GSTs into 2 subclasses—insect and MGST3. The tissue- and stage-expression patterns of the GSTs differed at the mRNA level. Further, the substrate specificities and kinetic constants of the cytosolic GSTs differed markedly at the protein level. The results of likelihood ratio tests provided strong evidence for positive selection in the delta class. The result of Bayes Empirical Bayes analysis identified 4 amino acid sites in the delta class as positive selection sites. These sites were located on the protein surface. Our findings will facilitate the elucidation of the molecular characteristics and evolutionary aspects of insect GST superfamily. PMID:25486043

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

    PubMed Central

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

    2015-01-01

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

  19. A unique geometry of the active site of angiotensin-converting enzyme consistent with structure-activity studies

    NASA Astrophysics Data System (ADS)

    Mayer, Dorica; Naylor, Christopher B.; Motoc, Ioan; Marshall, Garland R.

    1987-04-01

    Previous structure-activity studies of captopril and related active angiotensin-converting enzyme (ACE) inhibitors have led to the conclusion that the basic structural requirements for inhibition of ACE involve (a) a terminal carboxyl group; (b) an amido carbonyl group; and (c) different types of effective zinc (Zn) ligand functional groups. Such structural requirements common to a set of compounds acting at the same receptor have been used to define a pharmacophoric pattern of atoms or groups of atoms mutually oriented in space that is necessary for ACE inhibition from a stereochemical point of view. A unique pharmacophore model (within the resolution of approximately 0.15 Å) was observed using a method for systematic search of the conformational hyperspace available to the 28 structurally different molecules under study. The method does not assume a common molecular framework, and, therefore, allows comparison of different compounds that is independent of their absolute orientation. Consequently, by placing the carboxyl binding group, the binding site for amido carbonyl, and the Zn atom site in positions determined by ideal binding geometry with the inhibitors' functional groups, it was possible to clearly specify a geometry for the active site of ACE.

  20. LYN-activating mutations mediate antiestrogen resistance in estrogen receptor–positive breast cancer

    PubMed Central

    Schwarz, Luis J.; Fox, Emily M.; Balko, Justin M.; Garrett, Joan T.; Kuba, María Gabriela; Estrada, Mónica Valeria; González-Angulo, Ana María; Mills, Gordon B.; Red-Brewer, Monica; Mayer, Ingrid A.; Abramson, Vandana; Rizzo, Monica; Kelley, Mark C.; Meszoely, Ingrid M.; Arteaga, Carlos L.

    2014-01-01

    Estrogen receptor–positive (ER+) breast cancers adapt to hormone deprivation and become resistant to antiestrogen therapy. Here, we performed deep sequencing on ER+ tumors that remained highly proliferative after treatment with the aromatase inhibitor letrozole and identified a D189Y mutation in the inhibitory SH2 domain of the SRC family kinase (SFK) LYN. Evaluation of 463 breast tumors in The Cancer Genome Atlas revealed four LYN mutations, two of which affected the SH2 domain. In addition, LYN was upregulated in multiple ER+ breast cancer lines resistant to long-term estrogen deprivation (LTED). An RNAi-based kinome screen revealed that LYN is required for growth of ER+ LTED breast cancer cells. Kinase assays and immunoblot analyses of SRC substrates in transfected cells indicated that LYND189Y has higher catalytic activity than WT protein. Further, LYND189Y exhibited reduced phosphorylation at the inhibitory Y507 site compared with LYNWT. Other SH2 domain LYN mutants, E159K and K209N, also exhibited higher catalytic activity and reduced inhibitory site phosphorylation. LYND189Y overexpression abrogated growth inhibition by fulvestrant and/or the PI3K inhibitor BKM120 in 3 ER+ breast cancer cell lines. The SFK inhibitor dasatinib enhanced the antitumor effect of BKM120 and fulvestrant against estrogen-deprived ER+ xenografts but not LYND189Y-expressing xenografts. These results suggest that LYN mutations mediate escape from antiestrogens in a subset of ER+ breast cancers. PMID:25401474

  1. Position paper on active countermeasures for computer networks.

    SciTech Connect

    Van Randwyk, Jamie A.

    2003-07-01

    Computer security professionals have used passive network countermeasures for several years in order to secure computer networks. Passive countermeasures such as firewalls and intrusion detection systems are effective but their use alone is not enough to protect a network. Active countermeasures offer new ways of protecting a computer network. Corporations and government entities should adopt active network countermeasures as a means of protecting their computer networks.

  2. 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.

  3. cVEMP morphology changes with recording electrode position, but single motor unit activity remains constant.

    PubMed

    Rosengren, Sally M; Colebatch, James G; Borire, Adeniyi; Straumann, Dominik; Weber, Konrad P

    2016-04-15

    Cervical vestibular evoked myogenic potentials (cVEMPs) recorded over the lower quarter of the sternocleidomastoid (SCM) muscle in normal subjects may have opposite polarity to those recorded over the midpoint. It has thus been suggested that vestibular projections to the lower part of SCM might be excitatory rather than inhibitory. We tested the hypothesis that the SCM muscle receives both inhibitory and excitatory vestibular inputs. We recorded cVEMPs in 10 normal subjects with surface electrodes placed at multiple sites along the anterior (sternal) component of the SCM muscle. We compared several reference sites: sternum, ipsilateral and contralateral earlobes, and contralateral wrist. In five subjects, single motor unit responses were recorded at the upper, middle, and lower parts of the SCM muscle using concentric needle electrodes. The surface cVEMP had the typical positive-negative polarity at the midpoint of the SCM muscle. In all subjects, as the recording electrode was moved toward each insertion point, p13 amplitude became smaller and p13 latency increased, then the polarity inverted to a negative-positive waveform (n1-p1). Changing the reference site did not affect reflex polarity. There was a significant short-latency change in activity in 61/63 single motor units, and in each case this was a decrease or gap in firing, indicating an inhibitory reflex. Single motor unit recordings showed that the reflex was inhibitory along the entire SCM muscle. The cVEMP surface waveform inversion near the mastoid and sternal insertion points likely reflects volume conduction of the potential occurring with increasing distance from the motor point.

  4. Positive and negative reinforcement activate human auditory cortex.

    PubMed

    Weis, Tina; Puschmann, Sebastian; Brechmann, André; Thiel, Christiane M

    2013-01-01

    Prior studies suggest that reward modulates neural activity in sensory cortices, but less is known about punishment. We used functional magnetic resonance imaging and an auditory discrimination task, where participants had to judge the duration of frequency modulated tones. In one session correct performance resulted in financial gains at the end of the trial, in a second session incorrect performance resulted in financial loss. Incorrect performance in the rewarded as well as correct performance in the punishment condition resulted in a neutral outcome. The size of gains and losses was either low or high (10 or 50 Euro cent) depending on the direction of frequency modulation. We analyzed neural activity at the end of the trial, during reinforcement, and found increased neural activity in auditory cortex when gaining a financial reward as compared to gaining no reward and when avoiding financial loss as compared to receiving a financial loss. This was independent on the size of gains and losses. A similar pattern of neural activity for both gaining a reward and avoiding a loss was also seen in right middle temporal gyrus, bilateral insula and pre-supplemental motor area, here however neural activity was lower after correct responses compared to incorrect responses. To summarize, this study shows that the activation of sensory cortices, as previously shown for gaining a reward is also seen during avoiding a loss.

  5. Physical properties of naked DNA influence nucleosome positioning and correlate with transcription start and termination sites in yeast

    PubMed Central

    2011-01-01

    Background In eukaryotic organisms, DNA is packaged into chromatin structure, where most of DNA is wrapped into nucleosomes. DNA compaction and nucleosome positioning have clear functional implications, since they modulate the accessibility of genomic regions to regulatory proteins. Despite the intensive research effort focused in this area, the rules defining nucleosome positioning and the location of DNA regulatory regions still remain elusive. Results Naked (histone-free) and nucleosomal DNA from yeast were digested by microccocal nuclease (MNase) and sequenced genome-wide. MNase cutting preferences were determined for both naked and nucleosomal DNAs. Integration of their sequencing profiles with DNA conformational descriptors derived from atomistic molecular dynamic simulations enabled us to extract the physical properties of DNA on a genomic scale and to correlate them with chromatin structure and gene regulation. The local structure of DNA around regulatory regions was found to be unusually flexible and to display a unique pattern of nucleosome positioning. Ab initio physical descriptors derived from molecular dynamics were used to develop a computational method that accurately predicts nucleosome enriched and depleted regions. Conclusions Our experimental and computational analyses jointly demonstrate a clear correlation between sequence-dependent physical properties of naked DNA and regulatory signals in the chromatin structure. These results demonstrate that nucleosome positioning around TSS (Transcription Start Site) and TTS (Transcription Termination Site) (at least in yeast) is strongly dependent on DNA physical properties, which can define a basal regulatory mechanism of gene expression. PMID:21981773

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

    PubMed Central

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

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

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

    PubMed Central

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

    2010-01-01

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

  8. Quantitative test of the barrier nucleosome model for statistical positioning of nucleosomes up- and downstream of transcription start sites.

    PubMed

    Möbius, Wolfram; Gerland, Ulrich

    2010-08-19

    The positions of nucleosomes in eukaryotic genomes determine which parts of the DNA sequence are readily accessible for regulatory proteins and which are not. Genome-wide maps of nucleosome positions have revealed a salient pattern around transcription start sites, involving a nucleosome-free region (NFR) flanked by a pronounced periodic pattern in the average nucleosome density. While the periodic pattern clearly reflects well-positioned nucleosomes, the positioning mechanism is less clear. A recent experimental study by Mavrich et al. argued that the pattern observed in Saccharomyces cerevisiae is qualitatively consistent with a "barrier nucleosome model," in which the oscillatory pattern is created by the statistical positioning mechanism of Kornberg and Stryer. On the other hand, there is clear evidence for intrinsic sequence preferences of nucleosomes, and it is unclear to what extent these sequence preferences affect the observed pattern. To test the barrier nucleosome model, we quantitatively analyze yeast nucleosome positioning data both up- and downstream from NFRs. Our analysis is based on the Tonks model of statistical physics which quantifies the interplay between the excluded-volume interaction of nucleosomes and their positional entropy. We find that although the typical patterns on the two sides of the NFR are different, they are both quantitatively described by the same physical model with the same parameters, but different boundary conditions. The inferred boundary conditions suggest that the first nucleosome downstream from the NFR (the +1 nucleosome) is typically directly positioned while the first nucleosome upstream is statistically positioned via a nucleosome-repelling DNA region. These boundary conditions, which can be locally encoded into the genome sequence, significantly shape the statistical distribution of nucleosomes over a range of up to approximately 1,000 bp to each side.

  9. Mutation at positively selected positions in the binding site for HLA-C shows that KIR2DL1 is a more refined but less adaptable NK cell receptor than KIR2DL3.

    PubMed

    Hilton, Hugo G; Vago, Luca; Older Aguilar, Anastazia M; Moesta, Achim K; Graef, Thorsten; Abi-Rached, Laurent; Norman, Paul J; Guethlein, Lisbeth A; Fleischhauer, Katharina; Parham, Peter

    2012-08-01

    Through recognition of HLA class I, killer cell Ig-like receptors (KIR) modulate NK cell functions in human immunity and reproduction. Although a minority of HLA-A and -B allotypes are KIR ligands, HLA-C allotypes dominate this regulation, because they all carry either the C1 epitope recognized by KIR2DL2/3 or the C2 epitope recognized by KIR2DL1. The C1 epitope and C1-specific KIR evolved first, followed several million years later by the C2 epitope and C2-specific KIR. Strong, varying selection pressure on NK cell functions drove the diversification and divergence of hominid KIR, with six positions in the HLA class I binding site of KIR being targets for positive diversifying selection. Introducing each naturally occurring residue at these positions into KIR2DL1 and KIR2DL3 produced 38 point mutants that were tested for binding to 95 HLA- A, -B, and -C allotypes. Modulating specificity for HLA-C is position 44, whereas positions 71 and 131 control cross-reactivity with HLA-A*11:02. Dominating avidity modulation is position 70, with lesser contributions from positions 68 and 182. KIR2DL3 has lower avidity and broader specificity than KIR2DL1. Mutation could increase the avidity and change the specificity of KIR2DL3, whereas KIR2DL1 specificity was resistant to mutation, and its avidity could only be lowered. The contrasting inflexibility of KIR2DL1 and adaptability of KIR2DL3 fit with C2-specific KIR having evolved from C1-specific KIR, and not vice versa. Substitutions restricted to activating KIR all reduced the avidity of KIR2DL1 and KIR2DL3, further evidence that activating KIR function often becomes subject to selective attenuation.

  10. Differential Assembly of Catalytic Interactions within the Conserved Active Sites of Two Ribozymes

    PubMed Central

    Herschlag, Daniel

    2016-01-01

    Molecular recognition is central to biology and a critical aspect of RNA function. Yet structured RNAs typically lack the preorganization needed for strong binding and precise positioning. A striking example is the group I ribozyme from Tetrahymena, which binds its guanosine substrate (G) orders of magnitude slower than diffusion. Binding of G is also thermodynamically coupled to binding of the oligonucleotide substrate (S) and further work has shown that the transition from E•G to E•S•G accompanies a conformational change that allows G to make the active site interactions required for catalysis. The group I ribozyme from Azoarcus has a similarly slow association rate but lacks the coupled binding observed for the Tetrahymena ribozyme. Here we test, using G analogs and metal ion rescue experiments, whether this absence of coupling arises from a higher degree of preorganization within the Azoarcus active site. Our results suggest that the Azoarcus ribozyme forms cognate catalytic metal ion interactions with G in the E•G complex, interactions that are absent in the Tetrahymena E•G complex. Thus, RNAs that share highly similar active site architectures and catalyze the same reactions can differ in the assembly of transition state interactions. More generally, an ability to readily access distinct local conformational states may have facilitated the evolutionary exploration needed to attain RNA machines that carry out complex, multi-step processes. PMID:27501145

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

    PubMed

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

    2015-12-11

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

  12. Different positively selected sites at the gametophytic self-incompatibility pistil S-RNase gene in the Solanaceae and Rosaceae (Prunus, Pyrus, and Malus).

    PubMed

    Vieira, Jorge; Morales-Hojas, Ramiro; Santos, Raquel A M; Vieira, Cristina P

    2007-08-01

    In this work we perform a comparative study on the location of positively selected sites (those likely responsible for defining specificity differences) at the S-RNase gene, the pistil component of the gametophytic self-incompatibility system. For Plantaginaceae and Rosaceae (Prunus and Pyrus/Malus) this is the first study of this kind. A clear sign of positive selection was observed for 13, 17, and 27 amino acid sites in Solanaceae, Prunus, and Pyrus/Malus, respectively, using two different methodologies. In Plantaginaceae no clear positively selected sites were identified. Possible reasons for this result are discussed. Indirect experimental evidence suggests that the identified positively selected amino acid sites play a role in specificity determination. The percentage of positively selected sites is similar in Solanaceae and Rosaceae but the location of those sites is different.

  13. Global positioning system reoccupation of early triangulation sites - Tectonic deformation of the Southern Coast Ranges

    NASA Technical Reports Server (NTRS)

    Shen, Zheng-Kang; Jackson, David D.

    1993-01-01

    We study tectonic deformation in the Southern Coast Range, California. We use triangulation and astronomic azimuth data collected since 1875, trilateration since 1970, and global positioning system data collected from 1986 to 1987. Two modeling techniques have been used. An elastic block-fault model is applied to study the tectonic motion of the San Andreas Fault and the San Gregorio-Hosgri Fault. Station velocities are modeled to study regional deformations. Results show that the regional deformation is predominantly controlled by deep strike-slip motion along the San Andreas Fault, at a rate of 33 +/- 2 mm/yr. Deep slip along the San Gregorio-Hosgri Fault is about 0-4 mm/yr, assuming a locked suit to a depth of 20 km. Convergence normal to the San Andreas Fault in the Southern Coast Ranges is not greater than 0.02 microrad/yr.

  14. Extensive Evolutionary Changes in Regulatory Element Activity during Human Origins Are Associated with Altered Gene Expression and Positive Selection

    PubMed Central

    Fedrigo, Olivier; Babbitt, Courtney C.; Wortham, Matthew; Tewari, Alok K.; London, Darin; Song, Lingyun; Lee, Bum-Kyu; Iyer, Vishwanath R.; Parker, Stephen C. J.; Margulies, Elliott H.; Wray, Gregory A.; Furey, Terrence S.; Crawford, Gregory E.

    2012-01-01

    Understanding the molecular basis for phenotypic differences between humans and other primates remains an outstanding challenge. Mutations in non-coding regulatory DNA that alter gene expression have been hypothesized as a key driver of these phenotypic differences. This has been supported by differential gene expression analyses in general, but not by the identification of specific regulatory elements responsible for changes in transcription and phenotype. To identify the genetic source of regulatory differences, we mapped DNaseI hypersensitive (DHS) sites, which mark all types of active gene regulatory elements, genome-wide in the same cell type isolated from human, chimpanzee, and macaque. Most DHS sites were conserved among all three species, as expected based on their central role in regulating transcription. However, we found evidence that several hundred DHS sites were gained or lost on the lineages leading to modern human and chimpanzee. Species-specific DHS site gains are enriched near differentially expressed genes, are positively correlated with increased transcription, show evidence of branch-specific positive selection, and overlap with active chromatin marks. Species-specific sequence differences in transcription factor motifs found within these DHS sites are linked with species-specific changes in chromatin accessibility. Together, these indicate that the regulatory elements identified here are genetic contributors to transcriptional and phenotypic differences among primate species. PMID:22761590

  15. Characterization of Active Site Residues of Nitroalkane Oxidase†

    PubMed Central

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

    2010-01-01

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

  16. 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.

  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.

  18. Interaction of mining activities and aquatic environment: A review from Greek mine sites.

    NASA Astrophysics Data System (ADS)

    Vasileiou, Eleni; Kallioras, Andreas

    2016-04-01

    In Greece a significant amount of mineral and ore deposits have been recorded accompanied by large industrial interest and a long mining history. Today many active and/or abandoned mine sites are scattered within the country; while mining activities take place in different sites for exploiting various deposits (clay, limestone, slate, gypsum, kaolin, mixed sulphide ores (lead, zinc, olivine, pozzolan, quartz lignite, nickel, magnesite, aluminum, bauxite, gold, marbles etc). The most prominent recent ones are: (i) the lignite exploitation that is extended in the area of Ptolemais (Western Macedonia) and Megalopolis (Central Peloponnese); and (ii) the major bauxite deposits located in central Greece within the Parnassos-Ghiona geotectonic zone and on Euboea Island. In the latter area, significant ores of magnesite were exploited and mixed sulphide ores. Centuries of intensive mining exploitation and metallurgical treatment of lead-silver deposits in Greece, have also resulted in significant abandoned sites, such as the one in Lavrion. Mining activities in Lavrio, were initiated in ancient times and continued until the 1980s, resulting in the production of significant waste stockpiles deposited in the area, crucial for the local water resources. Ιn many mining sites, environmental pressures are also recorded after the mine closure to the aquatic environment, as the surface waters flow through waste dump areas and contaminated soils. This paper aims to the geospatial visualization of the mining activities in Greece, in connection to their negative (surface- and/or ground-water pollution; overpumping due to extensive dewatering practices) or positive (enhanced groundwater recharge; pit lakes, improvement of water budget in the catchment scale) impacts on local water resources.

  19. Distinguishing binders from false positives by free energy calculations: fragment screening against the flap site of HIV protease.

    PubMed

    Deng, Nanjie; Forli, Stefano; He, Peng; Perryman, Alex; Wickstrom, Lauren; Vijayan, R S K; Tiefenbrunn, Theresa; Stout, David; Gallicchio, Emilio; Olson, Arthur J; Levy, Ronald M

    2015-01-22

    Molecular docking is a powerful tool used in drug discovery and structural biology for predicting the structures of ligand-receptor complexes. However, the accuracy of docking calculations can be limited by factors such as the neglect of protein reorganization in the scoring function; as a result, ligand screening can produce a high rate of false positive hits. Although absolute binding free energy methods still have difficulty in accurately rank-ordering binders, we believe that they can be fruitfully employed to distinguish binders from nonbinders and reduce the false positive rate. Here we study a set of ligands that dock favorably to a newly discovered, potentially allosteric site on the flap of HIV-1 protease. Fragment binding to this site stabilizes a closed form of protease, which could be exploited for the design of allosteric inhibitors. Twenty-three top-ranked protein-ligand complexes from AutoDock were subject to the free energy screening using two methods, the recently developed binding energy analysis method (BEDAM) and the standard double decoupling method (DDM). Free energy calculations correctly identified most of the false positives (≥83%) and recovered all the confirmed binders. The results show a gap averaging ≥3.7 kcal/mol, separating the binders and the false positives. We present a formula that decomposes the binding free energy into contributions from the receptor conformational macrostates, which provides insights into the roles of different binding modes. Our binding free energy component analysis further suggests that improving the treatment for the desolvation penalty associated with the unfulfilled polar groups could reduce the rate of false positive hits in docking. The current study demonstrates that the combination of docking with free energy methods can be very useful for more accurate ligand screening against valuable drug targets.

  20. Structure-activity relationships of nonisomerizable derivatives of tamoxifen: importance of hydroxyl group and side chain positioning for biological activity.

    PubMed

    Murphy, C S; Parker, C J; McCague, R; Jordan, V C

    1991-03-01

    The antiestrogen tamoxifen [(Z)-1(p-beta-dimethylaminoethoxy-phenyl)-1,2-diphenylbut-1-ene] is an effective anticancer agent against estrogen receptor (ER)-positive breast cancer. The alkylaminoethane side chain is essential for antiestrogenic activity, but the potency of the antiestrogen can be increased by para hydroxylation of the phenyl ring on carbon 1 of but-1-ene. This compound, 4-hydroxytamoxifen, is a metabolite of tamoxifen and has a very high binding affinity for ER [J. Endocrinol. 75:305-316 (1977)] because the hydroxyl is located in the equivalent position as the 3-phenolic hydroxyl of 17 beta-estradiol. In this study, we have examined the relationship between the relative positions of the hydroxyl and the alkyl-aminoethane side chain and the pharmacological activity of the ligand. A fixed seven-membered ring derivative of the triphenylethylene was used to prevent isomerization. All compounds were tested, with and without 17 beta-estradiol, for their effects on the growth of estrogen-responsive T47D and MCF-7 human breast cancer cells in vitro. The growth of MDA-MB-231 ER-negative breast cancer cells was not affected by any of the compounds tested, at a concentration (1 microM) that had a profound estrogenic or antiestrogenic action in ER-positive cell lines. The relative binding affinity of the compounds was determined using rat uterine ER and was found to be consistent with the observed potencies in vitro. The compounds found to be antiestrogens in vitro were antiestrogenic against estradiol (0.08 micrograms daily) in the 3-day immature rat uterine weight test. All compounds were partial agonists in vivo. In general, the estrogenic and antiestrogenic results obtained in vivo were consistent with the potency estimates obtained with the breast cancer cells in vitro. The results of this extensive structure-activity relationship study demonstrate that the substitution for 4-hydroxytamoxifen appears to be optimal to produce a potent antiestrogen; all

  1. Structure–function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ

    PubMed Central

    Manuse, Sylvie; Jean, Nicolas L.; Guinot, Mégane; Lavergne, Jean-Pierre; Laguri, Cédric; Bougault, Catherine M.; VanNieuwenhze, Michael S.; Grangeasse, Christophe; Simorre, Jean-Pierre

    2016-01-01

    Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae, the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure–function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division. PMID:27346279

  2. Structure-function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ

    NASA Astrophysics Data System (ADS)

    Manuse, Sylvie; Jean, Nicolas L.; Guinot, Mégane; Lavergne, Jean-Pierre; Laguri, Cédric; Bougault, Catherine M.; Vannieuwenhze, Michael S.; Grangeasse, Christophe; Simorre, Jean-Pierre

    2016-06-01

    Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae, the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure-function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division.

  3. Propionibacterium acnes: Time-to-Positivity in Standard Bacterial Culture From Different Anatomical Sites

    PubMed Central

    Abdulmassih, Rasha; Makadia, Jina; Como, James; Paulson, Michelle; Min, Zaw; Bhanot, Nitin

    2016-01-01

    Background Propionibacterium acnes infections are likely under-recognized and underreported. This is partly because of low clinical suspicion, perceived non-pathogenicity, or lack of adequate culture incubation time. We conducted a study to assess the optimal incubation period to recover P. acnes from specimens acquired during the workup of suspected clinical infections. Methods A 5-year retrospective chart review was conducted between January 2010 and December 2014 at a single tertiary-care hospital. All patient cases from which P. acnes was recovered were included for analysis. Source of infection, antibiotic use, and culture time-to-positivity (TTP) were recorded. Results Implanted devices comprised the single most common source of P. acnes infection. In the majority of cases, P. acnes was the only organism identified. The mean incubation TTP for all isolates was 5.73 days. Conclusions Standard 5-day culture incubation periods are insufficient to recover P. acnes. As a result, P. acnes is likely a much more common etiology of a variety of clinical infections than previously reported. PMID:27829959

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

    PubMed

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

    2016-09-26

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

  5. Enhanced immunoPET of ALCAM-positive colorectal carcinoma using site-specific ⁶⁴Cu-DOTA conjugation.

    PubMed

    Tavaré, Richard; Wu, Wei H; Zettlitz, Kirstin A; Salazar, Felix B; McCabe, Katelyn E; Marks, James D; Wu, Anna M

    2014-10-01

    Activated leukocyte cell adhesion molecule (ALCAM) is an immunoglobulin superfamily cell adhesion molecule that is aberrantly expressed in a wide variety of human tumors, including melanoma, prostate cancer, breast cancer, colorectal carcinoma, bladder cancer and pancreatic adenocarcinoma. This wide spectrum of human malignancies makes ALCAM a prospective pan-cancer immunoPET target to aid in detection and diagnosis in multiple malignancies. In this study, we assess site-specific versus non-site-specific conjugation strategies for (64)Cu-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) immunoPET imaging of a fully human ALCAM cys-diabody (cDb) with a reduced linker length that retains its bivalent binding ability. ALCAM constructs with linker lengths of eight, five and three amino acids were produced to make true non-covalent site-specifically modified cDbs. Characterization by gel electrophoresis, size exclusion chromatography, flow cytometry and mass spectrometry of the various constructs was performed. To demonstrate the increased utility of targeting multiple malignancies expressing ALCAM, we compare the targeting of the site-specific versus non-site-specific conjugated cDbs to the human colorectal cancer xenograft LS174T. Interestingly, the conjugation strategy not only affects tumor targeting but also hepatic and renal uptake/clearance.

  6. Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis

    PubMed Central

    Martick, Monika; Scott, William G.

    2015-01-01

    SUMMARY Minimal hammerhead ribozymes have been characterized extensively by static and time-resolved crystallography as well as numerous biochemical analyses, leading to mutually contradictory mechanistic explanations for catalysis. We present the 2.2 Å resolution crystal structure of a full-length Schistosoma mansoni hammerhead ribozyme that permits us to explain the structural basis for its 1000-fold catalytic enhancement. The full-length hammerhead structure reveals how tertiary interactions occurring remotely from the active site prime this ribozyme for catalysis. G-12 and G-8 are positioned consistent with their previously suggested roles in acid-base catalysis, the nucleophile is aligned with a scissile phosphate positioned proximal to the A-9 phosphate, and previously unexplained roles of other conserved nucleotides become apparent within the context of a distinctly new fold that nonetheless accommodates the previous structural studies. These interactions permit us to explain the previously irreconcilable sets of experimental results in a unified, consistent, and unambiguous manner. PMID:16859740

  7. Simulation analysis of formycin 5?-monophosphate analog substrates in the ricin A-chain active site

    NASA Astrophysics Data System (ADS)

    Olson, Mark A.; Scovill, John P.; Hack, Dallas C.

    1995-06-01

    Ricin is an RNA N-glycosidase that hydrolyzes a single adenine base from a conserved loop of 28S ribosomal RNA, thus inactivating protein synthesis. Molecular-dynamics simulation methods are used to analyze the structural interactions and thermodynamics that govern the binding of formycin 5'-monophosphate (FMP) and several of its analogs to the active site of ricin A-chain. Simulations are carried out initiated from the X-ray crystal structure of the ricin-FMP complex with the ligand modeled as a dianion, monoanion and zwitterion. Relative changes in binding free energies are estimated for FMP analogs constructed from amino substitutions at the 2- and 2'-positions, and from hydroxyl substitution at the 2'-position.

  8. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

  9. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

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

  10. Orthogonal electrode catheter array for mapping of endocardial focal site of ventricular activation

    SciTech Connect

    Desai, J.M.; Nyo, H.; Vera, Z.; Seibert, J.A.; Vogelsang, P.J. )

    1991-04-01

    Precise location of the endocardial site of origin of ventricular tachycardia may facilitate surgical and catheter ablation of this arrhythmia. The endocardial catheter mapping technique can locate the site of ventricular tachycardia within 4-8 cm2 of the earliest site recorded by the catheter. This report describes an orthogonal electrode catheter array (OECA) for mapping and radiofrequency ablation (RFA) of endocardial focal site of origin of a plunge electrode paced model of ventricular activation in dogs. The OECA is an 8 F five pole catheter with four peripheral electrodes and one central electrode (total surface area 0.8 cm{sup 2}). In eight mongrel dogs, mapping was performed by arbitrarily dividing the left ventricle (LV) into four segments. Each segment was mapped with OECA to find the earliest segment. Bipolar and unipolar electrograms were obtained. The plunge electrode (not visible on fluoroscopy) site was identified by the earliest wave front arrival times of -30 msec or earlier at two or more electrodes (unipolar electrograms) with reference to the earliest recorded surface ECG (I, AVF, and V1). Validation of the proximity of the five electrodes of the OECA to the plunge electrode was performed by digital radiography and RFA. Pathological examination was performed to document the proximity of the OECA to the plunge electrode and also for the width, depth, and microscopic changes of the ablation. To find the segment with the earliest LV activation a total of 10 {plus minus} 3 (mean {plus minus} SD) positions were mapped. Mean arrival times at the two earlier electrodes were -39 {plus minus} 4 msec and -35 {plus minus} 3 msec. Digital radiography showed the plunge electrode to be within the area covered by all five electrodes in all eight dogs. The plunge electrode was within 1 cm2 area of the region of RFA in all eight dogs.

  11. Evidence of positive selection at codon sites localized in extracellular domains of mammalian CC motif chemokine receptor proteins

    PubMed Central

    2010-01-01

    Background CC chemokine receptor proteins (CCR1 through CCR10) are seven-transmembrane G-protein coupled receptors whose signaling pathways are known for their important roles coordinating immune system responses through targeted trafficking of white blood cells. In addition, some of these receptors have been identified as fusion proteins for viral pathogens: for example, HIV-1 strains utilize CCR5, CCR2 and CCR3 proteins to obtain cellular entry in humans. The extracellular domains of these receptor proteins are involved in ligand-binding specificity as well as pathogen recognition interactions. In mammals, the majority of chemokine receptor genes are clustered together; in humans, seven of the ten genes are clustered in the 3p21-24 chromosome region. Gene conversion events, or exchange of DNA sequence between genes, have been reported in chemokine receptor paralogs in various mammalian lineages, especially between the cytogenetically closely located pairs CCR2/5 and CCR1/3. Datasets of mammalian orthologs for each gene were analyzed separately to minimize the potential confounding impact of analyzing highly similar sequences resulting from gene conversion events. Molecular evolution approaches and the software package Phylogenetic Analyses by Maximum Likelihood (PAML) were utilized to investigate the signature of selection that has acted on the mammalian CC chemokine receptor (CCR) gene family. The results of neutral vs. adaptive evolution (positive selection) hypothesis testing using Site Models are reported. In general, positive selection is defined by a ratio of nonsynonymous/synonymous nucleotide changes (dN/dS, or ω) >1. Results Of the ten mammalian CC motif chemokine receptor sequence datasets analyzed, only CCR2 and CCR3 contain amino acid codon sites that exhibit evidence of positive selection using site based hypothesis testing in PAML. Nineteen of the twenty codon sites putatively indentified as likely to be under positive selection code for amino acid

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

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

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

    Code of Federal Regulations, 2011 CFR

    2011-01-01

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

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

    Code of Federal Regulations, 2013 CFR

    2013-01-01

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

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

    Code of Federal Regulations, 2012 CFR

    2012-01-01

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

  16. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2014 CFR

    2014-01-01

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

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

    SciTech Connect

    Teese, G.D.

    1995-09-28

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

  18. Facets of dynamic positive affect: differentiating joy, interest, and activation in the positive and negative affect schedule (PANAS).

    PubMed

    Egloff, Boris; Schmukle, Stefan C; Burns, Lawrence R; Kohlmann, Carl-Walter; Hock, Michael

    2003-09-01

    This article proposes the differentiation of Joy, Interest, and Activation in the Positive Affect (PA) scale of the Positive and Negative Affect Schedule (PANAS; D. Watson, L. A. Clark, & A. Tellegen, 1988). Study 1 analyzed the dynamic course of PA before, during, and after an exam and established the differentiation of the three facets. Study 2 used a multistate-multitrait analysis to confirm this structure. Studies 3-5 used success-failure experiences, speaking tasks, and feedback of exam results to further examine PA facets in affect-arousing settings. All studies provide convincing evidence for the benefit of differentiating three facets of PA in the PANAS: Joy, Interest, and Activation do have distinct and sometimes even opposite courses that make their separation meaningful and rewarding.

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

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Yellapragada, Bhavani Kumar

    2016-05-01

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

  2. Dynamically achieved active site precision in enzyme catalysis.

    PubMed

    Klinman, Judith P

    2015-02-17

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

  3. Detecting grizzly bear use of ungulate carcasses using global positioning system telemetry and activity data

    USGS Publications Warehouse

    Ebinger, Michael R.; Haroldson, Mark A.; van Manen, Frank T.; Costello, Cecily M; Bjornlie, Daniel D; Thompson, Daniel J.; Gunther, Kerry A.; Fortin, Jennifer K.; Teisberg, Justin E.; Pils, Shannon R; White, P J; Cain, Steven L; Cross, Paul C.

    2016-01-01

    Global positioning system (GPS) wildlife collars have revolutionized wildlife research. Studies of predation by free-ranging carnivores have particularly benefited from the application of location clustering algorithms to determine when and where predation events occur. These studies have changed our understanding of large carnivore behavior, but the gains have concentrated on obligate carnivores. Facultative carnivores, such as grizzly/brown bears (Ursus arctos), exhibit a variety of behaviors that can lead to the formation of GPS clusters. We combined clustering techniques with field site investigations of grizzly bear GPS locations (n = 732 site investigations; 2004–2011) to produce 174 GPS clusters where documented behavior was partitioned into five classes (large-biomass carcass, small-biomass carcass, old carcass, non-carcass activity, and resting). We used multinomial logistic regression to predict the probability of clusters belonging to each class. Two cross-validation methods—leaving out individual clusters, or leaving out individual bears—showed that correct prediction of bear visitation to large-biomass carcasses was 78–88%, whereas the false-positive rate was 18–24%. As a case study, we applied our predictive model to a GPS data set of 266 bear-years in the Greater Yellowstone Ecosystem (2002–2011) and examined trends in carcass visitation during fall hyperphagia (September–October). We identified 1997 spatial GPS clusters, of which 347 were predicted to be large-biomass carcasses. We used the clustered data to develop a carcass visitation index, which varied annually, but more than doubled during the study period. Our study demonstrates the effectiveness and utility of identifying GPS clusters associated with carcass visitation by a facultative carnivore.

  4. Detecting grizzly bear use of ungulate carcasses using global positioning system telemetry and activity data.

    PubMed

    Ebinger, Michael R; Haroldson, Mark A; van Manen, Frank T; Costello, Cecily M; Bjornlie, Daniel D; Thompson, Daniel J; Gunther, Kerry A; Fortin, Jennifer K; Teisberg, Justin E; Pils, Shannon R; White, P J; Cain, Steven L; Cross, Paul C

    2016-07-01

    Global positioning system (GPS) wildlife collars have revolutionized wildlife research. Studies of predation by free-ranging carnivores have particularly benefited from the application of location clustering algorithms to determine when and where predation events occur. These studies have changed our understanding of large carnivore behavior, but the gains have concentrated on obligate carnivores. Facultative carnivores, such as grizzly/brown bears (Ursus arctos), exhibit a variety of behaviors that can lead to the formation of GPS clusters. We combined clustering techniques with field site investigations of grizzly bear GPS locations (n = 732 site investigations; 2004-2011) to produce 174 GPS clusters where documented behavior was partitioned into five classes (large-biomass carcass, small-biomass carcass, old carcass, non-carcass activity, and resting). We used multinomial logistic regression to predict the probability of clusters belonging to each class. Two cross-validation methods-leaving out individual clusters, or leaving out individual bears-showed that correct prediction of bear visitation to large-biomass carcasses was 78-88 %, whereas the false-positive rate was 18-24 %. As a case study, we applied our predictive model to a GPS data set of 266 bear-years in the Greater Yellowstone Ecosystem (2002-2011) and examined trends in carcass visitation during fall hyperphagia (September-October). We identified 1997 spatial GPS clusters, of which 347 were predicted to be large-biomass carcasses. We used the clustered data to develop a carcass visitation index, which varied annually, but more than doubled during the study period. Our study demonstrates the effectiveness and utility of identifying GPS clusters associated with carcass visitation by a facultative carnivore.

  5. The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites.

    PubMed

    Chevalier, B S; Monnat, R J; Stoddard, B L

    2001-04-01

    Homing endonucleases, like restriction enzymes, cleave double-stranded DNA at specific target sites. The cleavage mechanism(s) utilized by LAGLIDADG endonucleases have been difficult to elucidate; their active sites are divergent, and only one low resolution cocrystal structure has been determined. Here we report two high resolution structures of the dimeric I-CreI homing endonuclease bound to DNA: a substrate complex with calcium and a product complex with magnesium. The bound metals in both complexes are verified by manganese anomalous difference maps. The active sites are positioned close together to facilitate cleavage across the DNA minor groove; each contains one metal ion bound between a conserved aspartate (Asp 20) and a single scissile phosphate. A third metal ion bridges the two active sites. This divalent cation is bound between aspartate residues from the active site of each subunit and is in simultaneous contact with the scissile phosphates of both DNA strands. A metal-bound water molecule acts as the nucleophile and is part of an extensive network of ordered water molecules that are positioned by enzyme side chains. These structures illustrate a unique variant of a two-metal endonuclease mechanism is employed by the highly divergent LAGLIDADG enzyme family.

  6. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    PubMed Central

    Lin, Xiaofeng; Ayrapetov, Marina K; Sun, Gongqin

    2005-01-01

    Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1) binds to ATP, and the other (M2) acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator. PMID:16305747

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

    PubMed Central

    Vallee, B L; Auld, D S

    1990-01-01

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

  8. What Factors Coordinate the Optimal Position of a Single Monitoring Well Down Gradient of a Hazardous Site?

    NASA Astrophysics Data System (ADS)

    Bode, F.; Nowak, W.

    2013-12-01

    Drinking-water well catchments include many sources for potential contaminations like gas stations, roads, or fields used for agriculture. Additionally, there are many contaminated sites that need to be monitored inside and outside drinking water catchments. Finding optimal positions of monitoring wells for such purposes is challenging because there are various parameters (and their uncertainties) that influence the reliability and optimality of a suggested monitoring location. For example, there may be uncertainty in the exact position of the contamination, in the source volume, in the direction of the velocity field which can vary in angle and absolute value, and in other parameters that describe, e.g., dispersion and decay. Many national regulations and UN guidelines suggest monitoring as measure of risk control, but make no statements how to asses or design monitoring under the fact of uncertainty. To obtain optimal positions of monitoring wells, a large body of recent studies uses formal optimization approaches. Our goal is to obtain a better system understanding at a fundamental process level for the one-on-one situation of a single monitoring well for a single monitoring target. This knowledge can be used for a better understanding of the optimization results in complex situations, and also to better guide and restrict optimization procedures by newly obtained export knowledge. In order to obtain fundamental statements regardless of specific simulation settings, we use an analytical model based on the 2D steady-state advection-dispersion equation to predict contaminant transport from the monitoring target. Monte Carlo simulation techniques are applied to represent parametric uncertainty. Thus, we can obtain maps of contaminant detection probability for all possible placements of the monitoring well. The optimal position is defined by the highest detection probability. First findings show that uncertainty in the spill location pushes the optimal monitoring

  9. Does Positive Selection Drive Transcription Factor Binding Site Turnover? A Test with Drosophila Cis-Regulatory Modules

    PubMed Central

    He, Bin Z.; Holloway, Alisha K.; Maerkl, Sebastian J.; Kreitman, Martin

    2011-01-01

    Transcription factor binding site(s) (TFBS) gain and loss (i.e., turnover) is a well-documented feature of cis-regulatory module (CRM) evolution, yet little attention has been paid to the evolutionary force(s) driving this turnover process. The predominant view, motivated by its widespread occurrence, emphasizes the importance of compensatory mutation and genetic drift. Positive selection, in contrast, although it has been invoked in specific instances of adaptive gene expression evolution, has not been considered as a general alternative to neutral compensatory evolution. In this study we evaluate the two hypotheses by analyzing patterns of single nucleotide polymorphism in the TFBS of well-characterized CRM in two closely related Drosophila species, Drosophila melanogaster and Drosophila simulans. An important feature of the analysis is classification of TFBS mutations according to the direction of their predicted effect on binding affinity, which allows gains and losses to be evaluated independently along the two phylogenetic lineages. The observed patterns of polymorphism and divergence are not compatible with neutral evolution for either class of mutations. Instead, multiple lines of evidence are consistent with contributions of positive selection to TFBS gain and loss as well as purifying selection in its maintenance. In discussion, we propose a model to reconcile the finding of selection driving TFBS turnover with constrained CRM function over long evolutionary time. PMID:21572512

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed

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

    2012-07-10

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

  12. Functional assignment to positively selected sites in the core type III effector RipG7 from Ralstonia solanacearum.

    PubMed

    Wang, Keke; Remigi, Philippe; Anisimova, Maria; Lonjon, Fabien; Kars, Ilona; Kajava, Andrey; Li, Chien-Hui; Cheng, Chiu-Ping; Vailleau, Fabienne; Genin, Stéphane; Peeters, Nemo

    2016-05-01

    The soil-borne pathogen Ralstonia solanacearum causes bacterial wilt in a broad range of plants. The main virulence determinants of R. solanacearum are the type III secretion system (T3SS) and its associated type III effectors (T3Es), translocated into the host cells. Of the conserved T3Es among R. solanacearum strains, the Fbox protein RipG7 is required for R. solanacearum pathogenesis on Medicago truncatula. In this work, we describe the natural ripG7 variability existing in the R. solanacearum species complex. We show that eight representative ripG7 orthologues have different contributions to pathogenicity on M. truncatula: only ripG7 from Asian or African strains can complement the absence of ripG7 in GMI1000 (Asian reference strain). Nonetheless, RipG7 proteins from American and Indonesian strains can still interact with M. truncatula SKP1-like/MSKa protein, essential for the function of RipG7 in virulence. This indicates that the absence of complementation is most likely a result of the variability in the leucine-rich repeat (LRR) domain of RipG7. We identified 11 sites under positive selection in the LRR domains of RipG7. By studying the functional impact of these 11 sites, we show the contribution of five positively selected sites for the function of RipG7CMR15 in M. truncatula colonization. This work reveals the genetic and functional variation of the essential core T3E RipG7 from R. solanacearum. This analysis is the first of its kind on an essential disease-controlling T3E, and sheds light on the co-evolutionary arms race between the bacterium and its hosts.

  13. LYN-activating mutations mediate antiestrogen resistance in estrogen receptor-positive breast cancer.

    PubMed

    Schwarz, Luis J; Fox, Emily M; Balko, Justin M; Garrett, Joan T; Kuba, María Gabriela; Estrada, Mónica Valeria; González-Angulo, Ana María; Mills, Gordon B; Red-Brewer, Monica; Mayer, Ingrid A; Abramson, Vandana; Rizzo, Monica; Kelley, Mark C; Meszoely, Ingrid M; Arteaga, Carlos L

    2014-12-01

    Estrogen receptor-positive (ER(+)) breast cancers adapt to hormone deprivation and become resistant to antiestrogen therapy. Here, we performed deep sequencing on ER(+) tumors that remained highly proliferative after treatment with the aromatase inhibitor letrozole and identified a D189Y mutation in the inhibitory SH2 domain of the SRC family kinase (SFK) LYN. Evaluation of 463 breast tumors in The Cancer Genome Atlas revealed four LYN mutations, two of which affected the SH2 domain. In addition, LYN was upregulated in multiple ER(+) breast cancer lines resistant to long-term estrogen deprivation (LTED). An RNAi-based kinome screen revealed that LYN is required for growth of ER(+) LTED breast cancer cells. Kinase assays and immunoblot analyses of SRC substrates in transfected cells indicated that LYN(D189Y) has higher catalytic activity than WT protein. Further, LYN(D189Y) exhibited reduced phosphorylation at the inhibitory Y507 site compared with LYN(WT). Other SH2 domain LYN mutants, E159K and K209N, also exhibited higher catalytic activity and reduced inhibitory site phosphorylation. LYN(D189Y) overexpression abrogated growth inhibition by fulvestrant and/or the PI3K inhibitor BKM120 in 3 ER(+) breast cancer cell lines. The SFK inhibitor dasatinib enhanced the antitumor effect of BKM120 and fulvestrant against estrogen-deprived ER(+) xenografts but not LYN(D189Y)-expressing xenografts. These results suggest that LYN mutations mediate escape from antiestrogens in a subset of ER(+) breast cancers.

  14. Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase.

    PubMed Central

    Stoughton, D M; Zapata, G; Picone, R; Vann, W F

    1999-01-01

    Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12-->Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in k(cat), K(m) and K(s) for CTP, which implicates Arg-12 in catalysis and substrate binding. PMID:10510306

  15. Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

    SciTech Connect

    Azzeh, Maysa; Honigman, Alik; Taraboulos, Albert; Rouvinski, Alexander; Wolf, Dana G. . E-mail: wolfd@md.huji.ac.il

    2006-10-10

    Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant ({delta}UL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the {delta}UL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or {delta}UL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites.

  16. Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity

    PubMed Central

    Panayiotou, Richard; Miralles, Francesc; Pawlowski, Rafal; Diring, Jessica; Flynn, Helen R; Skehel, Mark; Treisman, Richard

    2016-01-01

    The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A. DOI: http://dx.doi.org/10.7554/eLife.15460.001 PMID:27304076

  17. Sanctuary site leptomeningeal metastases in HER-2 positive breast cancer: A review in the era of trastuzumab.

    PubMed

    Kordbacheh, T; Law, W Y; Smith, I E

    2016-04-01

    The development of trastuzumab and other targeted systemic therapies has transformed the management of HER-2 positive breast cancers. However, as patients live longer and systemic therapies may not cross the blood brain barrier a rising number of patients are developing leptomeningeal metastases and brain metastases as a sanctuary site of disease. Intrathecal trastuzumab has been reported to treat these. We describe a breast cancer patient with HER-2 positive leptomeningeal disease in the spinal cord successfully treated with intrathecal trastuzumab and methotrexate, alongside systemic anti-HER-2 therapy and radiotherapy. We also review the literature to date on the efficacy and safety of intrathecal trastuzumab, and recent evidence suggesting that intrathecal trastuzumab passes via the blood brain barrier into the serum to achieve intravenous concentrations similar to that seen with systemic therapy alone. Overall, intrathecal trastuzumab appears to be a safe and often effective treatment for leptomeningeal metastases in HER-2 positive breast cancer. Ongoing phase I and II studies are required to determine optimum dosing schedules, validate CSF and CSF-to-serum pharmacokinetics, determine efficacy, and to assess the added benefits or disadvantages of prior radiotherapy and concomitant systemic therapy.

  18. Empathy is associated with dynamic change in prefrontal brain electrical activity during positive emotion in children

    PubMed Central

    Light, Sharee N.; Coan, James A.; Zahn-Waxler, Carolyn; Frye, Corrina; Goldsmith, H. Hill; Davidson, Richard J.

    2009-01-01

    Empathy is the combined ability to interpret the emotional states of others and experience resultant, related emotions. The relation between prefrontal electroencephalographic asymmetry and emotion in infants and children is well known. The relationship between positive emotion (assessed via parent-report), empathy (measured via observation) and second-by-second brain electrical activity (recorded during a pleasurable task) was investigated using a sample of 128 six to ten year olds. Contentment predicted increasing left-sided frontopolar activation (p<.05). Empathic concern and one form of positive empathy predicted increasing right-sided frontopolar activation (ps<.05). A second form of positive empathy predicted increasing left-sided dorsolateral activation (p<.05). This suggests that positive emotion and (negative and positive) empathy predict changes in prefrontal activity in children during a pleasurable task. PMID:19630903

  19. Enzyme-like specificity in zeolites: a unique site position in mordenite for selective carbonylation of methanol and dimethyl ether with CO.

    PubMed

    Boronat, Mercedes; Martínez-Sánchez, Cristina; Law, David; Corma, Avelino

    2008-12-03

    The mechanism of methanol carbonylation at different positions of zeolite MOR is investigated by quantum-chemical methods in order to discover which are the active sites that can selectively catalyze the desired reaction. It is shown that when methanol carbonylation competes with hydrocarbon formation, the first reaction occurs preferentially within 8MR channels. However, the unique selectivity for the carbonylation of methanol and dimethyl ether in mordenite is not only due to the size of the 8MR channel: neither process occurs equally at the two T3-O31 and T3-O33 positions. We show that only the T3-O33 positions are selective and that this selectivity is due to the unusual orientation of the methoxy group in relation to the 8MR channel (parallel to the cylinder axis). Only in this situation does the transition state for the attack of CO fit perfectly in the 8MR channel, while the reaction with methanol or DME is sterically impeded. This result explains why T3-O31, while also located in the 8MR channel of mordenite, is not as selective as the T3-O33 position and why ferrierite, although it contains 8MR channels, is less selective than mordenite. The competing effect of water is explained at the molecular level, and the molecular microkinetic reaction model has been established.

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

    PubMed

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

    2011-05-20

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

  1. A functional C-terminal TRAF3-binding site in MAVS participates in positive and negative regulation of the IFN antiviral response.

    PubMed

    Paz, Suzanne; Vilasco, Myriam; Werden, Steven J; Arguello, Meztli; Joseph-Pillai, Deshanthe; Zhao, Tiejun; Nguyen, Thi Lien-Anh; Sun, Qiang; Meurs, Eliane F; Lin, Rongtuan; Hiscott, John

    2011-06-01

    Recognition of viral RNA structures by the cytosolic sensor retinoic acid-inducible gene-I (RIG-I) results in the activation of signaling cascades that culminate with the generation of the type I interferon (IFN) antiviral response. Onset of antiviral and inflammatory responses to viral pathogens necessitates the regulated spatiotemporal recruitment of signaling adapters, kinases and transcriptional proteins to the mitochondrial antiviral signaling protein (MAVS). We previously demonstrated that the serine/threonine kinase IKKε is recruited to the C-terminal region of MAVS following Sendai or vesicular stomatitis virus (VSV) infection, mediated by Lys63-linked polyubiquitination of MAVS at Lys500, resulting in inhibition of downstream IFN signaling (Paz et al, Mol Cell Biol, 2009). In this study, we demonstrate that C-terminus of MAVS harbors a novel TRAF3-binding site in the aa450-468 region of MAVS. A consensus TRAF-interacting motif (TIM), 455-PEENEY-460, within this site is required for TRAF3 binding and activation of IFN antiviral response genes, whereas mutation of the TIM eliminates TRAF3 binding and the downstream IFN response. Reconstitution of MAVS(-/-) mouse embryo fibroblasts with a construct expressing a TIM-mutated version of MAVS failed to restore the antiviral response or block VSV replication, whereas wild-type MAVS reconstituted antiviral inhibition of VSV replication. Furthermore, recruitment of IKKε to an adjacent C-terminal site (aa 468-540) in MAVS via Lys500 ubiquitination decreased TRAF3 binding and protein stability, thus contributing to IKKε-mediated shutdown of the IFN response. This study demonstrates that MAVS harbors a functional C-terminal TRAF3-binding site that participates in positive and negative regulation of the IFN antiviral response.

  2. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    PubMed Central

    Holby, Edward F.; Taylor, Christopher D.

    2015-01-01

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O2 bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H2O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH bound structures have the highest calculated activity to date. PMID:25788358

  3. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    DOE PAGES

    Holby, Edward F.; Taylor, Christopher D.

    2015-03-19

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O₂ bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H₂O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH boundmore » structures have the highest calculated activity to date.« less

  4. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    SciTech Connect

    Holby, Edward F.; Taylor, Christopher D.

    2015-03-19

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O₂ bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H₂O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH bound structures have the highest calculated activity to date.

  5. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    NASA Astrophysics Data System (ADS)

    Holby, Edward F.; Taylor, Christopher D.

    2015-03-01

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O2 bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H2O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH bound structures have the highest calculated activity to date.

  6. 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)

  7. The Phylogeny and Active Site Design of Eukaryotic Copper-only Superoxide Dismutases

    DOE PAGES

    Peterson, Ryan L.; Galaleldeen, Ahmad; Villarreal, Johanna; ...

    2016-08-17

    In eukaryotes the bimetallic Cu/Zn superoxide dismutase (SOD) enzymes play important roles in the biology of reactive oxygen species by disproportionating superoxide anion. We reported that the fungal pathogen Candida albicans expresses a novel copper-only SOD, known as SOD5, that lacks the zinc cofactor and electrostatic loop (ESL) domain of Cu/Zn-SODs for substrate guidance. In spite of these abnormalities, C. albicans SOD5 can disproportionate superoxide at rates limited only by diffusion. Here we demonstrate that this curious copper-only SOD occurs throughout the fungal kingdom as well as in phylogenetically distant oomycetes or “pseudofungi” species. It is the only form ofmore » extracellular SOD in fungi and oomycetes, in stark contrast to the extracellular Cu/Zn-SODs of plants and animals. Through structural biology and biochemical approaches we demonstrate that these copper-only SODs have evolved with a specialized active site consisting of two highly conserved residues equivalent to SOD5 Glu-110 and Asp-113. The equivalent positions are zinc binding ligands in Cu/Zn-SODs and have evolved in copper-only SODs to control catalysis and copper binding in lieu of zinc and the ESL. Similar to the zinc ion in Cu/Zn-SODs, SOD5 Glu-110 helps orient a key copper-coordinating histidine and extends the pH range of enzyme catalysis. Furthermore, SOD5 Asp-113 connects to the active site in a manner similar to that of the ESL in Cu/Zn-SODs and assists in copper cofactor binding. Copper-only SODs are virulence factors for certain fungal pathogens; thus this unique active site may be a target for future anti-fungal strategies.« less

  8. The Phylogeny and Active Site Design of Eukaryotic Copper-only Superoxide Dismutases

    SciTech Connect

    Peterson, Ryan L.; Galaleldeen, Ahmad; Villarreal, Johanna; Taylor, Alexander B.; Cabelli, Diane E.; Hart, P. John; Culotta, Valeria C.

    2016-08-17

    In eukaryotes the bimetallic Cu/Zn superoxide dismutase (SOD) enzymes play important roles in the biology of reactive oxygen species by disproportionating superoxide anion. We reported that the fungal pathogen Candida albicans expresses a novel copper-only SOD, known as SOD5, that lacks the zinc cofactor and electrostatic loop (ESL) domain of Cu/Zn-SODs for substrate guidance. In spite of these abnormalities, C. albicans SOD5 can disproportionate superoxide at rates limited only by diffusion. Here we demonstrate that this curious copper-only SOD occurs throughout the fungal kingdom as well as in phylogenetically distant oomycetes or “pseudofungi” species. It is the only form of extracellular SOD in fungi and oomycetes, in stark contrast to the extracellular Cu/Zn-SODs of plants and animals. Through structural biology and biochemical approaches we demonstrate that these copper-only SODs have evolved with a specialized active site consisting of two highly conserved residues equivalent to SOD5 Glu-110 and Asp-113. The equivalent positions are zinc binding ligands in Cu/Zn-SODs and have evolved in copper-only SODs to control catalysis and copper binding in lieu of zinc and the ESL. Similar to the zinc ion in Cu/Zn-SODs, SOD5 Glu-110 helps orient a key copper-coordinating histidine and extends the pH range of enzyme catalysis. Furthermore, SOD5 Asp-113 connects to the active site in a manner similar to that of the ESL in Cu/Zn-SODs and assists in copper cofactor binding. Copper-only SODs are virulence factors for certain fungal pathogens; thus this unique active site may be a target for future anti-fungal strategies.

  9. Cloning and expression of acetylcholinesterase from Bungarus fasciatus venom. A new type of cooh-terminal domain; involvement of a positively charged residue in the peripheral site.

    PubMed

    Cousin, X; Bon, S; Duval, N; Massoulié, J; Bon, C

    1996-06-21

    As deduced from cDNA clones, the catalytic domain of Bungarus fasciatus venom acetylcholinesterase (AChE) is highly homologous to those of other AChEs. It is, however, associated with a short hydrophilic carboxyl-terminal region, containing no cysteine, that bears no resemblance to the alternative COOH-terminal peptides of the GPI-anchored molecules (H) or of other homomeric or heteromeric tailed molecules (T). Expression of complete and truncated AChE in COS cells showed that active hydrophilic monomers are produced and secreted in all cases, and that cleavage of a very basic 8-residue carboxyl-terminal fragment occurs upon secretion. The COS cells produced Bungarus AChE about 30 times more efficiently than an equivalent secreted monomeric rat AChE. The recombinant Bungarus AChE, like the natural venom enzyme, showed a distinctive ladder pattern in nondenaturing electrophoresis, probably reflecting a variation in the number of sialic acids. By mutagenesis, we showed that two differences (methionine instead of tyrosine at position 70; lysine instead of aspartate or glutamate at position 285) explain the low sensitivity of Bungarus AChE to peripheral site inhibitors, compared to the Torpedo or mammalian AChEs. These results illustrate the importance of both the aromatic and the charged residues, and the fact that peripheral site ligands (propidium, gallamine, D-tubocurarine, and fasciculin 2) interact with diverse subsets of residues.

  10. Effects of active site cleft residues on oligosaccharide binding, hydrolysis, and glycosynthase activities of rice BGlu1 and its mutants.

    PubMed

    Pengthaisong, Salila; Ketudat Cairns, James R

    2014-12-01

    Rice BGlu1 (Os3BGlu7) is a glycoside hydrolase family 1 β-glucosidase that hydrolyzes cellooligosaccharides with increasing efficiency as the degree of polymerization (DP) increases from 2 to 6, indicating six subsites for glucosyl residue binding. Five subsites have been identified in X-ray crystal structures of cellooligosaccharide complexes with its E176Q acid-base and E386G nucleophile mutants. X-ray crystal structures indicate that cellotetraose binds in a similar mode in BGlu1 E176Q and E386G, but in a different mode in the BGlu1 E386G/Y341A variant, in which glucosyl residue 4 (Glc4) interacts with Q187 instead of the eliminated phenolic group of Y341. Here, we found that the Q187A mutation has little effect on BGlu1 cellooligosaccharide hydrolysis activity or oligosaccharide binding in BGlu1 E176Q, and only slight effects on BGlu1 E386G glycosynthase activity. X-ray crystal structures showed that cellotetraose binds in a different position in BGlu1 E176Q/Y341A, in which it interacts directly with R178 and W337, and the Q187A mutation had little effect on cellotetraose binding. Mutations of R178 and W337 to A had significant and nonadditive effects on oligosaccharide hydrolysis by BGlu1, pNPGlc cleavage and cellooligosaccharide inhibition of BGlu1 E176Q and BGlu1 E386G glycosynthase activity. Hydrolysis activity was partially rescued by Y341 for longer substrates, suggesting stacking of Glc4 on Y341 stabilizes binding of cellooligosaccharides in the optimal position for hydrolysis. This analysis indicates that complex interactions between active site cleft residues modulate substrate binding and hydrolysis.

  11. 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.

  12. The position of prenylation of isoflavonoids and stilbenoids from legumes (Fabaceae) modulates the antimicrobial activity against Gram positive pathogens.

    PubMed

    Araya-Cloutier, Carla; den Besten, Heidy M W; Aisyah, Siti; Gruppen, Harry; Vincken, Jean-Paul

    2017-07-01

    The legume plant family (Fabaceae) is a potential source of antimicrobial phytochemicals. Molecular diversity in phytochemicals of legume extracts was enhanced by germination and fungal elicitation of seven legume species, as established by RP-UHPLC-UV-MS. The relationship between phytochemical composition, including different types of skeletons and substitutions, and antibacterial properties of extracts was investigated. Extracts rich in prenylated isoflavonoids and stilbenoids showed potent antibacterial activity against Listeria monocytogenes and methicillin-resistant Staphylococcus aureus at concentrations between 0.05 and 0.1% (w/v). Prenylated phenolic compounds were significantly (p<0.01) correlated with the antibacterial properties of the extracts. Furthermore, the position of the prenyl group within the phenolic skeleton also influenced the antibacterial activity. Overall, prenylated phenolics from legume seedlings can serve multiple purposes, e.g. as phytoestrogens they can provide health benefits and as natural antimicrobials they offer preservation of foods.

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

    PubMed

    Sun, XiaoYing; Li, Bo; Su, Dangsheng

    2014-09-28

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

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

    PubMed Central

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

    2016-01-01

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

  15. 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.

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2009-09-01

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

  18. Multiple, Ligand-Dependent Routes from the Active Site of Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Winn, Peter J.; Wade, Rebecca C.

    2012-02-13

    The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYP-membrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.

  19. Development of Novel Sugar Isomerases by Optimization of Active Sites in Phosphosugar Isomerases for Monosaccharides

    PubMed Central

    Yeom, Soo-Jin; Kim, Yeong-Su

    2013-01-01

    Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a d-ribose isomerase, an l-ribose isomerase, and an l-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases. PMID:23204422

  20. The effect of methyl-donated hydrogen bonding on active site conformations of hyaluronate lyase

    NASA Astrophysics Data System (ADS)

    Migues, Angela N.; Vergenz, Robert A.; Moore, Kevin B.

    2010-03-01

    Geometric evidence shows a val-A252 methyl-donated (MD) hydrogen bond (HB) in hyaluronate lyase (Streptococcus pneumoniae) interacts with nearby NH--O and OH--O HBs, distorting active-site helical structure. Results for model fragment A248-254 are based on experimental heavy atom positions with ab initio hydrogen atoms. The MDHB, with (H-O distance, donor-H-O angle) = (2.3å; 174^o), exhibits more favorable geometry than thr-A253 OH--O HB (1.8å; 170^o) to the same ala-249 C=O. Consequently, thr-253 N-H--O interaction is forced closer to lys-250 C=O than ala-249 C=O(2.6 versus 2.7å). A novel method has been developed to quantify the effects of atomic diplacements on motions of neighboring helices. A coordinate system was established to track the movement of specific residues and to ascertain the effect of such motions on active site conformations.

  1. Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity

    PubMed Central

    Dworeck, Tamara; Zimmermann, Martin

    2012-01-01

    Three different His-tagged, mutant forms of the fission yeast glutathione synthetase (GSH2) were derived by site-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5α and affinity purified in a two-step procedure. Analysis of enzyme activity showed that it was possible to shift the substrate specificity of GSH2 from Gly (km 0,19; wild-type) to β-Ala or Ser. One mutation (substitution of Ile471, Cy472 to Met and Val and Ala 485 and Thr486 to Leu and Pro) increased the affinity of GSH2 for β-Ala (km 0,07) and lowered the affinity for Gly (km 0,83), which is a characteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead the affinity of GSH2 for Ser (km 0,23) as a substrate, while affinity to Gly was preserved (km 0,12). This provides a new biosynthetic pathway for hydroxymethyl glutathione, which is known to be synthesized from glutathione and Ser in a reaction catalysed by carboxypeptidase Y. The reported findings provide further insight into how specific amino acids positioned in the GSH2 active site facilitate the recognition of different amino acid substrates, furthermore they support the evolutionary theory that homoglutathione synthetase evolved from glutathione synthetase by a single gene duplication event. PMID:23091597

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

    SciTech Connect

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

    1986-05-01

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

  3. Automated docking of maltose, 2-deoxymaltose, and maltotetraose into the soybean beta-amylase active site.

    PubMed

    Laederach, A; Dowd, M K; Coutinho, P M; Reilly, P J

    1999-11-01

    In this study, products and substrates were docked into the active site of beta-amylase using the simulated annealing algorithm AutoDock. Lowest-energy conformers reproduced known crystallographic atom positions within 0.4 to 0.8 A rmsd. Docking studies were carried out with both open and closed configurations of the beta-amylase mobile flap, a loop comprising residues 96 to 103. Ligands with two rings docked within the cleft near the active site when the flap was open, but those with four rings did not. The flap must be closed for alpha-maltotetraose to adopt a conformation allowing it to dock near the crystallographically determined subsites. The closed flap is necessary for productive but not for nonproductive binding, and therefore it plays a essential role in catalysis. The gain in total binding energy upon closing of the flap for alpha-maltose docked to subsites -2, -1 and +1, +2 is about 22 kcal/mol, indicating more favorable interactions are possible with the flap closed. Larger intermolecular interaction energies are observed for two alpha-maltose molecules docked to subsites -2, -1 and +1, +2 than for one alpha-maltotetraose molecule docked from subsites -2 to +2, suggesting that it is only upon cleavage of the alpha-1,4 linkage that optimal closed-flap binding can occur with the crytallographically determined enzyme structure.

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

    PubMed Central

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

    2015-01-01

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

  5. Nucleosomes Selectively Inhibit Cas9 Off-target Activity at a Site Located at the Nucleosome Edge.

    PubMed

    Hinz, John M; Laughery, Marian F; Wyrick, John J

    2016-11-25

    Nucleosomes affect Cas9 binding and activity at on-target sites, but their impact at off-target sites is unknown. To investigate how nucleosomes affect Cas9 cleavage at off-target sites in vitro, we used a single guide RNA (sgRNA) that has been previously shown to efficiently direct Cas9 cleavage at the edge of the strongly positioned 601 nucleosome. Our data indicate that single mismatches between the sgRNA and DNA target have relatively little effect on Cas9 cleavage of naked DNA substrates, but strongly inhibit cleavage of nucleosome substrates, particularly when the mismatch is in the sgRNA "seed" region. These findings indicate that nucleosomes may enhance Cas9 specificity by inhibiting cleavage of off-target sites at the nucleosome edge.

  6. 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.

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

    PubMed Central

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

    2013-01-01

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

  8. Using the Novel Method of Nonthermal Plasma To Add Cl Active Sites on Activated Carbon for Removal of Mercury from Flue Gas.

    PubMed

    Zhang, Bi; Zeng, Xiaobo; Xu, Ping; Chen, Juan; Xu, Yang; Luo, Guangqian; Xu, Minghou; Yao, Hong

    2016-11-01

    A new method using nonthermal plasma to add Cl active sites on activated carbon was proposed to improve the efficiency of activated carbon (AC) for removal of mercury from flue gas. The experiments were conducted via a lab-scale dielectric barrier discharge nonthermal plasma system and a vertical adsorption reactor. The results showed that the nonthermal plasma treatment with a small amount of Cl2 successfully added Cl active sites on AC and greatly increased the mercury removal efficiency of AC by chemisorption in a very short treatment time. The increase in Cl2 concentration for AC treatment promoted the efficiency of AC. The capacity of mercury adsorption positively correlated with the content of Cl2 for AC treatment, which depends on the number of Cl active sites on activated carbon. The treated AC maintained a high mercury removal efficiency within a temperature range of 30-210 °C. SO2 and H2O in flue gas inhibited the removal of mercury by AC, while HCl had a promotional effect. Scanning electron microscopy and X-ray photoelectron spectroscopy analysis indicated the chemisorption of mercury was attributed to the C-Cl groups generated on AC surfaces during Cl2 nonthermal plasma treatment. The C-Cl groups as active sites had strong adsorption energy for mercury, which converted elemental mercury to HgCl2.

  9. Synthesis and evaluation of M. tuberculosis salicylate synthase (MbtI) inhibitors designed to probe plasticity in the active site.

    PubMed

    Manos-Turvey, Alexandra; Cergol, Katie M; Salam, Noeris K; Bulloch, Esther M M; Chi, Gamma; Pang, Angel; Britton, Warwick J; West, Nicholas P; Baker, Edward N; Lott, J Shaun; Payne, Richard J

    2012-12-14

    Mycobacterium tuberculosis salicylate synthase (MbtI) catalyses the first committed step in the biosynthesis of mycobactin T, an iron-chelating siderophore essential for the virulence and survival of M. tuberculosis. Co-crystal structures of MbtI with members of a first generation inhibitor library revealed large inhibitor-induced rearrangements within the active site of the enzyme. This plasticity of the MbtI active site was probed via the preparation of a library of inhibitors based on a 2,3-dihydroxybenzoate scaffold with a range of substituted phenylacrylate side chains appended to the C3 position. Most compounds exhibited moderate inhibitory activity against the enzyme, with inhibition constants in the micromolar range, while several dimethyl ester variants possessed promising anti-tubercular activity in vitro.

  10. 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.

  11. Is carbon sequestration on post mining sites driven by earthworm activity?

    NASA Astrophysics Data System (ADS)

    Frouz, J.; Pizl, V.

    2009-04-01

    Carbon storage was measured in seven types of forest (alder, oak, lime, willow-birch, pine, spruce and larch) about 30 years old developed in on e large post mining site as split plot design. The carbon storage in soil wary substantial and represent 10-100% of carbon storage in aboveground wood biomass. Carbon storage in soil do not show any correlation with litter input but correlate significantly and positively with earthworm abundance, and micromorphological traces of earthworm activity. Field and laboratory microcosm experiment showed that earthworm mediated soil mixing support carbon storage in soil. Detailed study of soil aggregates created by worms and other forces indicated that worm aggregates contain much larger content of POC. This indicate that soil bioturbation by earthworms may significantly increase carbon storage in soil.

  12. Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure

    PubMed Central

    O'Farrell, Paul A.; Joshua-Tor, Leemor

    2006-01-01

    Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes. PMID:17007609

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

    PubMed

    Stec, Boguslaw

    2012-11-13

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a crucial enzyme in carbon fixation and the most abundant protein on earth. It has been studied extensively by biochemical and structural methods; however, the most essential activation step has not yet been described. Here, we describe the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO(2). We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O(2) and CO(2) bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO(2) defines an elusive, preactivation complex that contains a metal cation Mg(2+) surrounded by three H(2)O/OH molecules. Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism. Knowledge of all these elements is indispensable for engineering RuBisCO into a more efficient enzyme for crop enhancement or as a remedy to global warming.

  14. Changes in nucleosome position at transcriptional start sites of specific genes in Zea mays mediator of paramutation1 mutants

    PubMed Central

    Labonne, Jonathan D. J.; Dorweiler, Jane E.; McGinnis, Karen M.

    2013-01-01

    Nucleosomes facilitate compaction of DNA within the confines of the eukaryotic nucleus. This packaging of DNA and histone proteins must accommodate cellular processes, such as transcription and DNA replication. The repositioning of nucleosomes to facilitate cellular processes is likely regulated by several factors. In Zea mays, Mediator of paramutation1 (MOP1) has been demonstrated to be an epigenetic regulator of gene expression. Based on sequence orthology and mutant phenotypes, MOP1 is likely to function in an RNA-dependent pathway to mediate changes to chromatin. High-resolution microarrays were used to assay the distribution of nucleosomes across the transcription start sites (TSSs) of ~400 maize genes in wild type and mutant mop1–1 tissues. Analysis of nucleosome distribution in leaf, immature tassel and ear shoot tissues resulted in the identification of three genes showing consistent differences in nucleosome positioning and occupancy between wild type and mutant mop1–1. These specific changes in nucleosome distribution were located upstream as well as downstream of the TSS. No direct relationship between the specific changes in nucleosome distribution and transcription were observed through quantitative expression analysis in these tissues. In silico prediction suggests that nucleosome positioning is not dictated by intrinsic DNA sequence signals in the TSSs of two of the identified genes, suggesting a role for chromatin remodeling proteins in MOP1-mediated pathways. These results also indicate that MOP1 contributions to nucleosome position may be either separate from changes in gene expression, or cooperative with development and other levels of regulation in coordinating gene expression. PMID:23538550

  15. Changes in nucleosome position at transcriptional start sites of specific genes in Zea mays mediator of paramutation1 mutants.

    PubMed

    Labonne, Jonathan D J; Dorweiler, Jane E; McGinnis, Karen M

    2013-04-01

    Nucleosomes facilitate compaction of DNA within the confines of the eukaryotic nucleus. This packaging of DNA and histone proteins must accommodate cellular processes, such as transcription and DNA replication. The repositioning of nucleosomes to facilitate cellular processes is likely regulated by several factors. In Zea mays, Mediator of paramutation1 (MOP1) has been demonstrated to be an epigenetic regulator of gene expression. Based on sequence orthology and mutant phenotypes, MOP1 is likely to function in an RNA-dependent pathway to mediate changes to chromatin. High-resolution microarrays were used to assay the distribution of nucleosomes across the transcription start sites (TSSs) of ~400 maize genes in wild type and mutant mop1-1 tissues. Analysis of nucleosome distribution in leaf, immature tassel and ear shoot tissues resulted in the identification of three genes showing consistent differences in nucleosome positioning and occupancy between wild type and mutant mop1-1. These specific changes in nucleosome distribution were located upstream as well as downstream of the TSS. No direct relationship between the specific changes in nucleosome distribution and transcription were observed through quantitative expression analysis in these tissues. In silico prediction suggests that nucleosome positioning is not dictated by intrinsic DNA sequence signals in the TSSs of two of the identified genes, suggesting a role for chromatin remodeling proteins in MOP1-mediated pathways. These results also indicate that MOP1 contributions to nucleosome position may be either separate from changes in gene expression, or cooperative with development and other levels of regulation in coordinating gene expression.

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

    PubMed Central

    Mlýnský, Vojtěch; Šponer, Jiří

    2014-01-01

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

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

    DTIC Science & Technology

    2013-08-01

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

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

    PubMed Central

    2015-01-01

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

  19. 76 FR 30696 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-26

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

  20. 76 FR 24871 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-03

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

  1. Kinetic and structural evaluation of selected active site mutants of the Aspergillus fumigatus KDNase (sialidase).

    PubMed

    Yeung, Juliana H F; Telford, Judith C; Shidmoossavee, Fahimeh S; Bennet, Andrew J; Taylor, Garry L; Moore, Margo M

    2013-12-23

    Aspergillus fumigatus is an airborne fungal pathogen. We previously cloned and characterized an exo-sialidase from A. fumigatus and showed that it preferred 2-keto-3-deoxynononic acid (KDN) as a substrate to N-acetylneuraminic acid (Neu5Ac). The purpose of this study was to investigate the structure-function relationships of critical catalytic site residues. Site-directed mutagenesis was used to create three mutant recombinant enzymes: the catalytic nucleophile (Y358H), the general acid/base catalyst (D84A), and an enlargement of the binding pocket to attempt to accommodate the N-acetyl group of Neu5Ac (R171L). Crystal structures for all enzymes were determined. The D84A mutation had an effect in decreasing the activity of AfKDNase that was stronger than that of the same mutation in the structurally similar sialidase from the bacterium Micromonospora viridifaciens. These data suggest that the catalytic acid is more important in the reaction of AfKDNase and that catalysis is less dependent on nucleophilic or electrostatic stabilization of the developing positive charge at the transition state for hydrolysis. Removal of the catalytic nucleophile (Y358H) significantly lowered the activity of the enzyme, but this mutant remained a retaining glycosidase as demonstrated by nuclear magnetic resonance spectroscopic analysis. This is a novel finding that has not been shown with other sialidases. Kinetic activity measured at pH 5.2 revealed that R171L had higher activity on a Neu5Ac-based substrate than wild-type KDNase; hence, leucine in place of arginine in the binding pocket improved catalysis toward Neu5Ac substrates. Hence, whether a sialidase is primarily a KDNase or a neuraminidase is due in part to the presence of an amino acid that creates a steric clash with the N-acetyl group.

  2. The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.

    PubMed

    Ogden, S; Haggerty, D; Stoner, C M; Kolodrubetz, D; Schleif, R

    1980-06-01

    The locations of DNA binding by the proteins involved with positive and negative regulation of transcription initiation of the L-arabinose operon in Escherichia coli have been determined by the DNase I protection method. Two cyclic AMP receptor protein sites were found, at positions -78 to -107 and -121 to -146, an araC protein--arabinose binding site was found at position -40 to -78, and an araC protein-fucose binding site was found at position -106 to -144. These locations, combined with in vivo data on induction of the two divergently oriented arabinose promoters, suggest the following regulatory mechanism: induction of the araBAD operon occurs when cyclic AMP receptor protein, araC protein, and RNA polymerase are all present and able to bind to DNA. Negative regulation is accomplished by the repressing form of araC protein binding to a site in the regulatory region such that it stimultaneously blocks access of cyclic AMP receptor protein to two sites on the DNA, one site of which serves each of the two promoters. Thus, from a single operator site, the negative regulator represses the two outwardly oriented ara promoters. This regulatory mechanism explains the known positive and negative regulatory properties of the ara promoters.

  3. Influence of pelvis position on the activation of abdominal and hip flexor muscles.

    PubMed

    Workman, J Chad; Docherty, David; Parfrey, Kevin C; Behm, David G

    2008-09-01

    A pelvic position has been sought that optimizes abdominal muscle activation while diminishing hip flexor activation. Thus, the objective of the study was to investigate the effect of pelvic position and the Janda sit-up on trunk muscle activation. Sixteen male volunteers underwent electromyographic (EMG) testing of their abdominal and hip flexor muscles during a supine isometric double straight leg lift (DSLL) with the feet held approximately 5 cm above a board. The second exercise (Janda sit-up) was a sit-up action where participants simultaneously contracted the hamstrings and the abdominal musculature while holding an approximately 45 degrees angle at the knee. Root mean square surface electromyography was calculated for the Janda sit-up and DSLL under 3 pelvic positions: anterior, neutral, and posterior pelvic tilt. The selected muscles were the upper and lower rectus abdominis (URA, LRA), external obliques, lower abdominal stabilizers (LAS), rectus femoris, and biceps femoris. The Janda sit-up position demonstrated the highest URA and LRA activation and the lowest rectus femoris activation. The Janda sit-up and the posterior tilt were significantly greater (p < 0.01 and p < 0.05, respectively) than the anterior tilt for the URA and LRA muscles. Activation levels of the URA and LRA in neutral pelvis were significantly (p < 0.01 and p < 0.05, respectively) less than the Janda sit-up position, but not significantly different from the posterior tilt. No significant differences in EMG activity were found for the external obliques or LAS. No rectus femoris differences were found in the 3 pelvis positions. The results of this study indicate that pelvic position had a significant effect on the activation of selected trunk and hip muscles during isometric exercise, and the activation of the biceps femoris during the Janda sit-up reduced the activation of the rectus femoris while producing high levels of activation of the URA and LRA.

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

    PubMed

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

    2015-09-01

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

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

    PubMed

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

    1993-06-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2013-03-01

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

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

    PubMed Central

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

    1997-01-01

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

  9. 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.

  10. LXR activation inhibits chemokine-induced CD4-positive lymphocyte migration.

    PubMed

    Walcher, Daniel; Vasic, Dusica; Heinz, Philipp; Bach, Helga; Durst, Renate; Hausauer, Angelina; Hombach, Vinzenz; Marx, Nikolaus

    2010-07-01

    Migration of CD4-positive lymphocytes into the vessel wall is a critical step in atherogenesis. Recent data suggest that CD4-positive lymphocytes express the nuclear transcription factors Liver-X-Receptor (LXR) alpha and beta with an effect of LXR activators on TH1-cytokine release from these cells. However, the role of LXR in lymphocyte migration remains currently unexplored. Therefore, the present study investigated whether LXR activation might modulate chemokine-induced migration of these cells. Stimulation of CD4-positive lymphocytes with SDF-1 leads to a 2.5 +/- 0.8-fold increase in cell migration (P < 0.05; n = 12). Pretreatment of cells with the LXR activator T0901317 reduces this effect in a concentration-dependent manner to a maximal 0.9 +/- 0.4-fold induction at 1 micromol/L T0901317 (P < 0.05 compared to SDF-1-treated cells; n = 12). Similar results were obtained with the LXR activator GW3965. The effect of LXR activators on CD4-positive lymphocyte migration was mediated through an early inhibition of chemokine-induced PI-3 kinase activity as determined by PI-3 kinase activity assays. Downstream, T0901317 inhibited activation of the small GTPase Rac and phosphorylation of the myosin light chain (MLC). Moreover, LXR activator treatment reduced f-actin formation as well as ICAM3 translocation to the uropod of the cell, thus interfering with two important steps in T cell migration. Transfection of CD4-positive lymphocytes with LXRalpha/beta siRNA abolished T0901317 inhibitory effect on MLC phosphorylation and ICAM3 translocation. LXR activation by T0901317 or GW3965 inhibits chemokine-induced migration of CD4-positive lymphocytes. Given the crucial importance of chemokine-induced T cell migration in early atherogenesis, LXR activators may be promising tools to modulate this effect.

  11. Positivity effect in healthy aging in observational but not active feedback-learning.

    PubMed

    Bellebaum, Christian; Rustemeier, Martina; Daum, Irene

    2012-01-01

    The present study investigated the impact of healthy aging on the bias to learn from positive or negative performance feedback in observational and active feedback learning. In active learning, a previous study had already shown a negative learning bias in healthy seniors older than 75 years, while no bias was found for younger seniors. However, healthy aging is accompanied by a 'positivity effect', a tendency to primarily attend to stimuli with positive valence. Based on recent findings of dissociable neural mechanisms in active and observational feedback learning, the positivity effect was hypothesized to influence older participants' observational feedback learning in particular. In two separate experiments, groups of young (mean age 27) and older participants (mean age 60 years) completed an observational or active learning task designed to differentially assess positive and negative learning. Older but not younger observational learners showed a significant bias to learn better from positive than negative feedback. In accordance with previous findings, no bias was found for active learning. This pattern of results is discussed in terms of differences in the neural underpinnings of active and observational learning from performance feedback.

  12. Can positive matrix factorization help to understand patterns of organic trace gases at the continental Global Atmosphere Watch site Hohenpeissenberg?

    NASA Astrophysics Data System (ADS)

    Leuchner, M.; Gubo, S.; Schunk, C.; Wastl, C.; Kirchner, M.; Menzel, A.; Plass-Dülmer, C.

    2015-02-01

    From the rural Global Atmosphere Watch (GAW) site Hohenpeissenberg in the pre-alpine area of southern Germany, a data set of 24 C2-C8 non-methane hydrocarbons over a period of 7 years was analyzed. Receptor modeling was performed by positive matrix factorization (PMF) and the resulting factors were interpreted with respect to source profiles and photochemical aging. Differing from other studies, no direct source attribution was intended because, due to chemistry along transport, mass conservation from source to receptor is not given. However, at remote sites such as Hohenpeissenberg, the observed patterns of non-methane hydrocarbons can be derived from combinations of factors determined by PMF. A six-factor solution showed high stability and the most plausible results. In addition to a biogenic and a background factor of very stable compounds, four additional anthropogenic factors were resolved that could be divided into two short- and two long-lived patterns from evaporative sources/natural gas leakage and incomplete combustion processes. The volume or mass contribution at the site over the entire period was, in decreasing order, from the following factor categories: background, gas leakage and long-lived evaporative, residential heating and long-lived combustion, short-lived evaporative, short-lived combustion, and biogenic. The importance with respect to reactivity contribution was generally in reverse order, with the biogenic and the short-lived combustion factors contributing most. The seasonality of the factors was analyzed and compared to results of a simple box model using constant emissions and the photochemical decay calculated from the measured annual cycles of OH radicals and ozone. Two of the factors, short-lived combustion and gas leakage/long-lived evaporative, showed winter/summer ratios of about 9 and 7, respectively, as expected from constant source estimations. Contrarily, the short-lived evaporative emissions were about 3 times higher in summer

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

    DOE PAGES

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

    2016-11-29

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

  14. 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

  15. Effect of Knee Position on Quadriceps Muscle Force Steadiness and Activation Strategies

    PubMed Central

    Krishnan, Chandramouli; Allen, Eric J.; Williams, Glenn N.

    2010-01-01

    Introduction This study investigated the effect of knee position on quadriceps force steadiness and activation strategies. Methods Quadriceps force steadiness was evaluated in twenty-two volunteers at two knee positions by testing their ability to regulate submaximal force. Muscle activation strategies were studied in both time and frequency domains using surface electromyography. Results Quadriceps force fluctuations and the associated agonist and antagonist activity were significantly higher at 90° than at 30° of flexion (P < 0.05). The quadriceps median frequency recorded at 30° was significantly higher than at 90° of flexion (P < 0.05). Regression analyses revealed that force steadiness was related to quadriceps activation and median frequency (P < 0.001), but not to hamstring coactivation (P > 0.05). Discussion The results indicate that knee position significantly affects quadriceps force steadiness and activation strategies. This finding may have important implications for designing a force control testing protocol and interpreting test results. PMID:21404288

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

    PubMed Central

    2015-01-01

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

  17. Analysis of the activity of virus internal ribosome entry site in silkworm Bombyx mori.

    PubMed

    Ye, Lupeng; Zhuang, Lanfang; Li, Jisheng; You, Zhengying; Liang, Jianshe; Wei, Hao; Lin, Jianrong; Zhong, Boxiong

    2013-07-01

    Internal ribosome entry site (IRES) has been widely used in genetic engineering; however, the application in silkworm (Bombyx mori) has hardly been reported. In this study, the biological activity of partial sequence of Encephalomyocarditis virus (EMCV) IRES, Rhopalosiphum padi virus (RhPV) IRES, and the hybrid of IRES of EMCV and RhPV were investigated in Spodoptera frugiperda (Sf9) cell line and silkworm tissues. The hybrid IRES of EMCV and RhPV showed more effective than EMCV IRES or RhPV IRES in promoting downstream gene expression in insect and silkworm. The activities of all IRESs in middle silk gland of silkworm were higher than those in the fat body and posterior silk gland. The hybrid IRES of EMCV and RhPV was integrated into silkworm genome by transgenic technology to test biological activity of IRES. Each of the positive transgenic individuals had significant expression of report gene EGFP. These results suggested that IRES has a potential to be used in the genetic engineering research of silkworm.

  18. 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…

  19. Activity of the positive and negative reinforcement motivation systems and baseline arterial blood pressure in humans.

    PubMed

    Aftanas, L I; Sidorova, P V; Pavlov, S V; Makhnev, V P; Korenek, V V; Reva, N V; Amstislavskaya, T G

    2008-10-01

    The aim of the present work was to identify possible associations between individual balances in the activity of the positive and negative reinforcement motivation systems using a method based on emotional modulation of the startle reaction (EMSR) by motivationally significant emotionally positive and negative contextual visual stimuli and measures of cardiovascular system activity. Studies were performed using healthy males (mean age 30.29 +/- 9.8 years) with normal and first-episode excessive increases in arterial blood pressure (systolic blood pressure to greater than 140 mmHg, diastolic to greater than 90 mmHg). Cluster analysis of EMSR data identified groups of individuals with different activity profiles for the positive and negative reinforcement systems. Groups of subjects with changes in the balance of activity towards a lower level of positive reinforcement system activity (smaller startle reflexes to positive contextual stimuli) or a higher level of negative reinforcement system activity (larger startle reactions to threatening contextual stimuli) showed significantly greater baseline SBP and DBP. The possible mechanisms of the modulatory influences of the balance of system activities on autonomic vascular regulatory processes are discussed.

  20. 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

  1. Coupling chemical transport model source attributions with positive matrix factorization: application to two IMPROVE sites impacted by wildfires.

    PubMed

    Sturtz, Timothy M; Schichtel, Bret A; Larson, Timothy V

    2014-10-07

    Source contributions to total fine particle carbon predicted by a chemical transport model (CTM) were incorporated into the positive matrix factorization (PMF) receptor model to form a receptor-oriented hybrid model. The level of influence of the CTM versus traditional PMF was varied using a weighting parameter applied to an object function as implemented in the Multilinear Engine (ME-2). The methodology provides the ability to separate features that would not be identified using PMF alone, without sacrificing fit to observations. The hybrid model was applied to IMPROVE data taken from 2006 through 2008 at Monture and Sula Peak, Montana. It was able to separately identify major contributions of total carbon (TC) from wildfires and minor contributions from biogenic sources. The predictions of TC had a lower cross-validated RMSE than those from either PMF or CTM alone. Two unconstrained, minor features were identified at each site, a soil derived feature with elevated summer impacts and a feature enriched in sulfate and nitrate with significant, but sporadic contributions across the sampling period. The respective mean TC contributions from wildfires, biogenic emissions, and other sources were 1.18, 0.12, and 0.12 ugC/m(3) at Monture and 1.60, 0.44, and 0.06 ugC/m(3) at Sula Peak.

  2. Situational Motivation and Perceived Intensity: Their Interaction in Predicting Changes in Positive Affect from Physical Activity

    PubMed Central

    Guérin, Eva; Fortier, Michelle S.

    2012-01-01

    There is evidence that affective experiences surrounding physical activity can contribute to the proper self-regulation of an active lifestyle. Motivation toward physical activity, as portrayed by self-determination theory, has been linked to positive affect, as has the intensity of physical activity, especially of a preferred nature. The purpose of this experimental study was to examine the interaction between situational motivation and intensity [i.e., ratings of perceived exertion (RPE)] in predicting changes in positive affect following an acute bout of preferred physical activity, namely, running. Fourty-one female runners engaged in a 30-minute self-paced treadmill run in a laboratory context. Situational motivation for running, pre- and post-running positive affect, and RPE were assessed via validated self-report questionnaires. Hierarchical regression analyses revealed a significant interaction effect between RPE and introjection (P < .05) but not between RPE and identified regulation or intrinsic motivation. At low levels of introjection, the influence of RPE on the change in positive affect was considerable, with higher RPE ratings being associated with greater increases in positive affect. The implications of the findings in light of SDT principles as well as the potential contingencies between the regulations and RPE in predicting positive affect among women are discussed. PMID:22778914

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

    PubMed

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

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

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

    PubMed

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

    2012-07-10

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

  5. 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.

  6. Structure and Reactivity of the Phosphotriesterase Active Site

    DTIC Science & Technology

    2002-01-01

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

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

    PubMed

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

    2012-02-06

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

  8. Breadth and Intensity of Youth Activity Involvement as Contexts for Positive Development

    ERIC Educational Resources Information Center

    Rose-Krasnor, Linda; Busseri, Michael A.; Willoughby, Teena; Chalmers, Heather

    2006-01-01

    Research has linked youth activity involvement to positive development. However, past studies have confounded at least two separable dimensions of involvement: breadth (number of activities) and intensity (participation frequency). Theory and the limited available evidence suggest that these dimensions may make independent contributions to…

  9. Positive Behavior Interventions and Support in a Physical Activity Summer Camp

    ERIC Educational Resources Information Center

    Hinton, Vanessa; Buchanan, Alice M.

    2015-01-01

    This purpose of this study was to investigate the implementation of positive behavior interventions and support (PBIS) in a summer camp. The camp provided physical activity opportunities to underserved children attending a summer program at a local, rural public school. Certified physical education teachers led activity stations. Participants in…

  10. Physical Activity and Psychological Benefits. International Society of Sport Psychology Position Statement.

    ERIC Educational Resources Information Center

    Physician and Sportsmedicine, 1992

    1992-01-01

    International Society of Sport Psychology clarifies the psychological benefits of physical activity, noting the positive relationship between physical activity level and mental health. Exercise can reduce anxiety, decrease depression levels, reduce neuroticism and anxiety, reduce stress, and have beneficial emotional effects for both sexes across…

  11. Alignment of Hands-On STEM Engagement Activities with Positive STEM Dispositions in Secondary School Students

    ERIC Educational Resources Information Center

    Christensen, Rhonda; Knezek, Gerald; Tyler-Wood, Tandra

    2015-01-01

    This study examines positive dispositions reported by middle school and high school students participating in programs that feature STEM-related activities. Middle school students participating in school-to-home hands-on energy monitoring activities are compared to middle school and high school students in a different project taking part in…

  12. QM/MM calculations of kinetic isotope effects in the chorismate mutase active site.

    PubMed

    Martí, Sergio; Moliner, Vincent; Tuñón, Iñaki; Williams, Ian H

    2003-02-07

    Kinetic isotope effects have been computed for the Claisen rearrangement of chorismate to prephenate in aqueous solution and in the active site of chorismate mutase from B. subtilus. These included primary 13C and 18O and secondary 3H effects for substitutions at the bond-making and bond-breaking positions. The initial structures of the putative stationary points on the potential energy surface, required for the calculations of isotope effects using the CAMVIB/CAMISO programs, have been selected from hybrid QM/MM molecular dynamical simulations using the DYNAMO program. Refinement of the reactant complex and transition-state structures has been carried out by means of AM1/CHARMM24/TIP3P calculations using the GRACE program, with full gradient relaxation of the position of > 5200 atoms for the enzymic simulations, and with a box containing 711 water molecules for the corresponding reaction in aqueous solution. Comparison of these results, and of gas phase calculations, with experimental data has shown that the chemical rearrangement is largely rate-determining for the enzyme mechanism. Inclusion of the chorismate conformational pre-equilibrium step in the modelled kinetic scheme leads to better agreement between recent experimental data and theoretical predictions. These results provide new information on an important enzymatic transformation, and the key factors responsible for the kinetics of its molecular mechanism are clarified. Treatment of the enzyme and/or solvent environment by means of a large and flexible model is absolutely essential for prediction of kinetic isotope effects.

  13. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

    PubMed

    Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  14. Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

    PubMed Central

    Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I.; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity. PMID:24278284

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

    SciTech Connect

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

    2014-01-03

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

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

    PubMed Central

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

    2006-01-01

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

  17. Is spinal excitability of the triceps surae mainly affected by muscle activity or body position?

    PubMed

    Cattagni, T; Martin, A; Scaglioni, G

    2014-06-15

    The aim of this study was to determine how muscle activity and body orientation contribute to the triceps surae spinal transmission modulation, when moving from a sitting to a standing position. Maximal Hoffmann-reflex (Hmax) and motor potential (Mmax) were evoked in the soleus (SOL), medial and lateral gastrocnemius in 10 male subjects and in three conditions, passive sitting, active sitting and upright standing, with the same SOL activity in active sitting and upright standing. Moreover volitional wave (V) was evoked in the two active conditions (i.e., active sitting and upright standing). The results showed that SOL Hmax/Mmax was lower in active sitting than in passive sitting, while for the gastrocnemii it was not significantly altered. For the three plantar flexors, Hmax/Mmax was lower in upright standing than in active sitting, whereas V/Mmax was not modulated. SOL H-reflex is therefore affected by the increase in muscle activity and change in body orientation, while, in the gastrocnemii, it was only affected by a change in posture. In conclusion, passing from a sitting to a standing position affects the Hmax/Mmax of the whole triceps surae, but the mechanisms responsible for this change differ among the synergist muscles. The V/Mmax does not change when upright stance is assumed. This means that the increased inhibitory activity in orthostatic position is compensated by an increased excitatory inflow to the α-motoneurons of central and/or peripheral origin.

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

    PubMed

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

    2006-03-01

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

  19. Elm1 kinase activates the spindle position checkpoint kinase Kin4

    PubMed Central

    Caydasi, Ayse Koca; Kurtulmus, Bahtiyar; Orrico, Maria I.L.; Hofmann, Astrid; Ibrahim, Bashar

    2010-01-01

    Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck–associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4. PMID:20855503

  20. Elm1 kinase activates the spindle position checkpoint kinase Kin4.

    PubMed

    Caydasi, Ayse Koca; Kurtulmus, Bahtiyar; Orrico, Maria I L; Hofmann, Astrid; Ibrahim, Bashar; Pereira, Gislene

    2010-09-20

    Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck-associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.

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

    PubMed

    Stanne, Tara M; Rudenko, Gloria

    2010-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  3. The Demethylase JMJD2C Localizes to H3K4me3-Positive Transcription Start Sites and Is Dispensable for Embryonic Development

    PubMed Central

    Pedersen, Marianne Terndrup; Agger, Karl; Laugesen, Anne; Johansen, Jens V.; Cloos, Paul A. C.; Christensen, Jesper

    2014-01-01

    The histone demethylase JMJD2C, also known as KDM4C/GASC1, has activity against methylated H3K9 and H3K36 and is amplified and/or overexpressed in human cancers. By the generation of Jmjd2c knockout mice, we demonstrate that loss of Jmjd2c is compatible with cellular proliferation, embryonic stem cell (ESC) self-renewal, and embryonic development. Moreover, we report that JMJD2C localizes to H3K4me3-positive transcription start sites in both primary cells and in the human carcinoma KYSE150 cell line containing an amplification of the JMJD2C locus. Binding is dependent on the double Tudor domain of JMJD2C, which recognizes H3K4me3 but not H4K20me2/me3 in vitro, showing a binding specificity different from that of the double Tudor domains of JMJD2A and JMJD2B. Depletion of JMJD2C in KYSE150 cells has a modest effect on H3K9me3 and H3K36me3 levels but impairs proliferation and leads to deregulated expression of a subset of target genes involved in cell cycle progression. Taking these findings together, we show that JMJD2C is targeted to H3K4me3-positive transcription start sites, where it can contribute to transcriptional regulation, and report that the putative oncogene JMJD2C generally is not required for cellular proliferation or embryonic development. PMID:24396064

  4. 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

  5. Adaptability and Prediction of Anticipatory Muscular Activity Parameters to Different Movements in the Sitting Position.

    PubMed

    Chikh, Soufien; Watelain, Eric; Faupin, Arnaud; Pinti, Antonio; Jarraya, Mohamed; Garnier, Cyril

    2016-08-01

    Voluntary movement often causes postural perturbation that requires an anticipatory postural adjustment to minimize perturbation and increase the efficiency and coordination during execution. This systematic review focuses specifically on the relationship between the parameters of anticipatory muscular activities and movement finality in sitting position among adults, to study the adaptability and predictability of anticipatory muscular activities parameters to different movements and conditions in sitting position in adults. A systematic literature search was performed using PubMed, Science Direct, Web of Science, Springer-Link, Engineering Village, and EbscoHost. Inclusion and exclusion criteria were applied to retain the most rigorous and specific studies, yielding 76 articles, Seventeen articles were excluded at first reading, and after the application of inclusion and exclusion criteria, 23 were retained. In a sitting position, central nervous system activity precedes movement by diverse anticipatory muscular activities and shows the ability to adapt anticipatory muscular activity parameters to the movement direction, postural stability, or charge weight. In addition, these parameters could be adapted to the speed of execution, as found for the standing position. Parameters of anticipatory muscular activities (duration, order, and amplitude of muscle contractions constituting the anticipatory muscular activity) could be used as a predictive indicator of forthcoming movement. In addition, this systematic review may improve methodology in empirical studies and assistive technology for people with disabilities.

  6. Crystal structure of the ectoine hydroxylase, a snapshot of the active site.

    PubMed

    Höppner, Astrid; Widderich, Nils; Lenders, Michael; Bremer, Erhard; Smits, Sander H J

    2014-10-24

    Ectoine and its derivative 5-hydroxyectoine are compatible solutes that are widely synthesized by bacteria to cope physiologically with osmotic stress. They also serve as chemical chaperones and maintain the functionality of macromolecules. 5-Hydroxyectoine is produced from ectoine through a stereo-specific hydroxylation, an enzymatic reaction catalyzed by the ectoine hydroxylase (EctD). The EctD protein is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily and is evolutionarily well conserved. We studied the ectoine hydroxylase from the cold-adapted marine ultra-microbacterium Sphingopyxis alaskensis (Sa) and found that the purified SaEctD protein is a homodimer in solution. We determined the SaEctD crystal structure in its apo-form, complexed with the iron catalyst, and in a form that contained iron, the co-substrate 2-oxoglutarate, and the reaction product of EctD, 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the EctD active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-Hydroxyectoine, however, is coordinated by EctD in manner different from that found in high affinity solute receptor proteins operating in conjunction with microbial import systems for ectoines. Our crystallographic analysis provides a detailed view into the active site of the ectoine hydroxylase and exposes an intricate network of interactions between the enzyme and its ligands that collectively ensure the hydroxylation of the ectoine substrate in a position- and stereo-specific manner.

  7. Lateral flow urine lipoarabinomannan assay for detecting active tuberculosis in Hiv-positive adults

    PubMed Central

    Shah, Maunank; Hanrahan, Colleen; Wang, Zhuo Yu; Dendukuri, Nandini; Lawn, Stephen D; Denkinger, Claudia M; Steingart, Karen R

    2016-01-01

    Background Rapid detection of tuberculosis (TB) among people living with human immunodeficiency virus (HIV) is a global health priority. HIV-associated TB may have different clinical presentations and is challenging to diagnose. Conventional sputum tests have reduced sensitivity in HIV-positive individuals, who have higher rates of extrapulmonary TB compared with HIV-negative individuals. The lateral flow urine lipoarabinomannan assay (LF-LAM) is a new, commercially available point-of-care test that detects lipoarabinomannan (LAM), a lipopolysaccharide present in mycobacterial cell walls, in people with active TB disease. Objectives To assess the accuracy of LF-LAM for the diagnosis of active TB disease in HIV-positive adults who have signs and symptoms suggestive of TB (TB diagnosis).To assess the accuracy of LF-LAM as a screening test for active TB disease in HIV-positive adults irrespective of signs and symptoms suggestive of TB (TB screening). Search methods We searched the following databases without language restriction on 5 February 2015: the Cochrane Infectious Diseases Group Specialized Register; MEDLINE (PubMed,1966); EMBASE (OVID, from 1980); Science Citation Index Expanded (SCI-EXPANDED, from 1900), Conference Proceedings Citation Index-Science (CPCI-S, from 1900), and BIOSIS Previews (from 1926) (all three using the Web of Science platform; MEDION; LILACS (BIREME, from 1982); SCOPUS (from 1995); the metaRegister of Controlled Trials (mRCT); the search portal of the World Health Organization International Clinical Trials Registry Platform (WHO ICTRP); and ProQuest Dissertations & Theses A&l (from 1861). Selection criteria Eligible study types included randomized controlled trials, cross-sectional studies, and cohort studies that determined LF-LAM accuracy for TB against a microbiological reference standard (culture or nucleic acid amplification test from any body site). A higher quality reference standard was one in which two or more specimen types were

  8. Transcriptionally active immediate-early protein of pseudorabies virus binds to specific sites on class II gene promoters.

    PubMed Central

    Cromlish, W A; Abmayr, S M; Workman, J L; Horikoshi, M; Roeder, R G

    1989-01-01

    In the presence of partially purified pseudorabies virus immediate-early protein, multiple sites of DNase I protection were observed on the adenovirus major late and human hsp 70 promoters. Southwestern (DNA-protein blot) analysis demonstrated that the immediate-early protein bound directly to the sequences contained in these sites. These sequences share only limited homology, differ in their affinities for the immediate-early protein, and are located at different positions on these two promoters. In addition, the site-specific binding of a temperature-sensitive immediate-early protein was eliminated by the same heat treatment which eliminates its transcriptional activating function, whereas the binding of the wild-type protein was unaffected by heat treatment. Thus, site-specific binding requires a functionally active immediate-early protein. Furthermore, immediate-early-protein-dependent in vitro transcription from the major late promoter was preferentially inhibited by oligonucleotides which are homologous to the high-affinity binding sites on the major late or hsp 70 promoters. These observations suggest that transcriptional stimulation by the immediate-early protein involves binding to cis-acting elements. Images PMID:2539489

  9. Alignment of Hands-on STEM Engagement Activities with Positive STEM Dispositions in Secondary School Students

    NASA Astrophysics Data System (ADS)

    Christensen, Rhonda; Knezek, Gerald; Tyler-Wood, Tandra

    2015-12-01

    This study examines positive dispositions reported by middle school and high school students participating in programs that feature STEM-related activities. Middle school students participating in school-to-home hands-on energy monitoring activities are compared to middle school and high school students in a different project taking part in activities such as an after-school robotics program. Both groups are compared and contrasted with a third group of high school students admitted at the eleventh grade to an academy of mathematics and science. All students were assessed using the same science, technology, engineering and mathematics (STEM) dispositions instrument. Findings indicate that the after-school group whose participants self-selected STEM engagement activities, and the self-selected academy of mathematics and science group, each had highly positive STEM dispositions comparable to those of STEM professionals, while a subset of the middle school whole-classroom energy monitoring group that reported high interest in STEM as a career, also possessed highly positive STEM dispositions comparable to the STEM Professionals group. The authors conclude that several different kinds of hands-on STEM engagement activities are likely to foster or maintain positive STEM dispositions at the middle school and high school levels, and that these highly positive levels of dispositions can be viewed as a target toward which projects seeking to interest mainstream secondary students in STEM majors in college and STEM careers, can hope to aspire. Gender findings regarding STEM dispositions are also reported for these groups.

  10. 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...

  11. 75 FR 71677 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-24

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

  12. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...). (e) For all asbestos-containing waste material received, the owner or operator of the active waste... 40 Protection of Environment 9 2013-07-01 2013-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an...

  13. Electromyographic activity of preterm newborns in the kangaroo position: a cohort study

    PubMed Central

    Miranda, Rafael Moura; Cabral Filho, José Eulálio; Diniz, Kaísa Trovão; Souza Lima, Geisy Maria; Vasconcelos, Danilo de Almeida

    2014-01-01

    Objective To compare the electromyographic activity of preterm newborns placed in the kangaroo position with the activity of newborns not placed in this position. Design A cohort study. Setting A Kangaroo Unit sector and a Nursery sector in a secondary and tertiary care at a mother-child hospital in Recife, Brazil. Participants Preterm infants of gestational age 27–34 weeks (n=38) and term infants (n=39). Primary and secondary outcome measures Surface electromyography was used to investigate muscle activity in the brachial biceps at rest. 3 groups were designed: (1) preterm newborns in the kangaroo position (PT-KAN), where the newborn remains in a vertical position, lying face down, with limbs flexed, dressed in light clothes, maintaining skin-to-skin contact with the adult's thorax. Her electromyographic activity was recorded at 0 h (immediately before starting this position), and then at 48 h after the beginning of the position (but newborns were kept in the kangaroo position for 8–12 h per day) and at term equivalent age (40±1 weeks); (2) preterm newborns not in the kangaroo position (PT-NKAN), in which measurements were made at 0 h and 48 h; and (3) term newborns (T), in which measurements were made at 24 h of chronological age. Results The Root Mean Square (RMS) values showed significant differences among groups (F(5,108)=56.69; p<0.001). The multiple comparisons showed that RMS was greater at 48 h compared to 0 h in the preterm group in the kangaroo position, but not in the group not submitted in the kangaroo position. The RMS in the term equivalent aged group in the kangaroo position was also greater when compared with those in the term group. Conclusions The kangaroo position increases electromyographic activity in the brachial biceps of preterm newborns and those who have reached the age equivalent to term. PMID:25351598

  14. Physically adsorbed fullerene layer on positively charged sites on zinc oxide cathode affords efficiency enhancement in inverted polymer solar cell.

    PubMed

    Cheng, Yu-Shan; Liao, Sih-Hao; Li, Yi-Lun; Chen, Show-An

    2013-07-24

    We present a novel idea for overcoming the drawback of poor contact between the ZnO cathode and active layer interface in an inverted polymer solar cell (i-PSC), simply by incorporating an electron-acceptor self-assembled monolayer (SAM)--tetrafluoroterephthalic acid (TFTPA)--on the ZnO cathode surface to create an electron-poor surface of TFTPA on ZnO. The TFTPA molecules on ZnO are anchored on the ZnO surface by reacting its carboxyl groups with hydroxyl groups on the ZnO surface, such that the tetrafluoroterephthalate moieties lay on the surface with plane-on electron-poor benzene rings acting as positive charge centers. Upon coating a layer of fullerenes on top of it, the fullerene molecules can be physically adsorbed by Coulombic interaction and facilitate a promoted electron collection from the bulk. The active layer is composed of the mid bandgap polymer poly(3-hexylthiophene) (P3HT) or low bandgap polymer, poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b']dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl) carbonyl]thieno[3,4-b]thiophenediyl

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

    PubMed

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

    2003-05-13

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

  16. Structure of protein O-mannose kinase reveals a unique active site architecture

    PubMed Central

    Zhu, Qinyu; Venzke, David; Walimbe, Ameya S; Anderson, Mary E; Fu, Qiuyu; Kinch, Lisa N; Wang, Wei; Chen, Xing; Grishin, Nick V; Huang, Niu; Yu, Liping; Dixon, Jack E; Campbell, Kevin P; Xiao, Junyu

    2016-01-01

    The ‘pseudokinase’ SgK196 is a protein O-mannose kinase (POMK) that catalyzes an essential phosphorylation step during biosynthesis of the laminin-binding glycan on α-dystroglycan. However, the catalytic mechanism underlying this activity remains elusive. Here we present the crystal structure of Danio rerio POMK in complex with Mg2+ ions, ADP, aluminum fluoride, and the GalNAc-β3-GlcNAc-β4-Man trisaccharide substrate, thereby providing a snapshot of the catalytic transition state of this unusual kinase. The active site of POMK is established by residues located in non-canonical positions and is stabilized by a disulfide bridge. GalNAc-β3-GlcNAc-β4-Man is recognized by a surface groove, and the GalNAc-β3-GlcNAc moiety mediates the majority of interactions with POMK. Expression of various POMK mutants in POMK knockout cells further validated the functional requirements of critical residues. Our results provide important insights into the ability of POMK to function specifically as a glycan kinase, and highlight the structural diversity of the human kinome. DOI: http://dx.doi.org/10.7554/eLife.22238.001 PMID:27879205

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

    SciTech Connect

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

    2016-08-17

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

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

    DOE PAGES

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

    2016-08-17

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

  19. Size-resolved source apportionment of ambient particles by positive matrix factorization at Gosan background site in East Asia

    NASA Astrophysics Data System (ADS)

    Han, J. S.; Moon, K. J.; Lee, S. J.; Kim, Y. J.; Ryu, S. Y.; Cliff, S. S.; Yi, S. M.

    2006-01-01

    Size- and time-resolved aerosol samples were collected using an eight-stage Davis rotating unit for monitoring (DRUM) sampler from 29 March to 29 May in 2002 at Gosan, Jeju Island, Korea, which is one of the representative background sites in East Asia. These samples were analyzed using synchrotron X-ray fluorescence for 3-h average concentrations of 19 elements consisting of S, Si, Al, Fe, Ca, Cl, Cu, Zn, Ti, K, Mn, Pb, Ni, V, Se, As, Rb, Cr, Br. The size-resolved data sets were then analyzed using the positive matrix factorization (PMF) technique in order to identify possible sources and estimate their contribution to particulate matter mass. PMF analysis uses the uncertainty of the measured data to provide an optimal weighting. Fifteen sources were resolved in eight size ranges (0.07~12 μm) and included continental soil, local soil, sea salt, biomass/biofuel burning, coal combustion, oil heating furnace, residual oil fired boiler, municipal incineration, nonferrous metal source, ferrous metal source, gasoline vehicle, diesel vehicle, copper smelter and volcanic emission. PMF analysis of size-resolved source contributions showed that natural sources represented by local soil, sea salt and continental soil contributed about 79% to the predicted primary particulate matter (PM) mass in the coarse size range (1.15~12 μm). On the other hand, anthropogenic sources such as coal combustion and biomass/biofuel burning contributed about 60% in the fine size range (0.56~2.5 μm). The diesel vehicle source contributed the most in the ultra-fine size range (0.07~0.56 μm) and was responsible for about 52% of the primary PM mass.

  20. 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.

  1. Recurrent disruption of the Imu splice donor site in t(14;18) positive lymphomas: a potential molecular basis for aberrant downstream class switch recombination.

    PubMed

    Ruminy, Philippe; Jardin, Fabrice; Penther, Dominique; Picquenot, Jean-Michel; Parmentier, Françoise; Buchonnet, Gérard; Bertrand, Philippe; Tilly, Hervé; Bastard, Christian

    2007-08-01

    t(14;18) positive lymphomas are mature germinal center B-cell neoplasms. In agreement with this cellular origin, most have somatically mutated immunoglobulin variable genes and the IGH@ locus has almost always been reorganized by class switch recombination (CSR). However, contrasting with normal B-cells, a majority of cases still express an IgM while the constant genes are normally rearranged only on the non-productive allele. Concurrently, aberrant intra-allelic junctions involving downstream switch regions, with a lack of engagement of the switch mu (Smu), often accumulate on the functional alleles, suggesting some recurrent CSR perturbation during the onset of the disease. To clarify these surprising observations, we addressed the accessibility of the Smu to the CSR machinery in a large series of patients by characterizing the mutations that are expected to accumulate at this place upon CSR activation. Our data indicate that the Smu is mutated in a large majority of cases, often on both alleles, indicating that these cells usually reach a differentiation stage where CSR is activated and where this region remains accessible. Interestingly, we also identified a significant cluster of mutations at the splicing donor site of the first exon of the Smu germline transcripts, on the functional allele. This location suggests a possible relation with CSR perturbations in lymphoma and the clustering points to a probable mechanism of selection. In conclusion, our data suggest that an acquired mutation at the splicing donor site of the Smu transcripts may participate in the selection of lymphoma cells and play a significant role during the onset of the disease.

  2. American College of Sports Medicine position stand. Exercise and physical activity for older adults.

    PubMed

    Chodzko-Zajko, Wojtek J; Proctor, David N; Fiatarone Singh, Maria A; Minson, Christopher T; Nigg, Claudio R; Salem, George J; Skinner, James S

    2009-07-01

    The purpose of this Position Stand is to provide an overview of issues critical to understanding the importance of exercise and physical activity in older adult populations. The Position Stand is divided into three sections: Section 1 briefly reviews the structural and functional changes that characterize normal human aging, Section 2 considers the extent to which exercise and physical activity can influence the aging process, and Section 3 summarizes the benefits of both long-term exercise and physical activity and shorter-duration exercise programs on health and functional capacity. Although no amount of physical activity can stop the biological aging process, there is evidence that regular exercise can minimize the physiological effects of an otherwise sedentary lifestyle and increase active life expectancy by limiting the development and progression of chronic disease and disabling conditions. There is also emerging evidence for significant psychological and cognitive benefits accruing from regular exercise participation by older adults. Ideally, exercise prescription for older adults should include aerobic exercise, muscle strengthening exercises, and flexibility exercises. The evidence reviewed in this Position Stand is generally consistent with prior American College of Sports Medicine statements on the types and amounts of physical activity recommended for older adults as well as the recently published 2008 Physical Activity Guidelines for Americans. All older adults should engage in regular physical activity and avoid an inactive lifestyle.

  3. Purification, enzymatic properties, and active site environment of a novel manganese(III)-containing acid phosphatase.

    PubMed

    Sugiura, Y; Kawabe, H; Tanaka, H; Fujimoto, S; Ohara, A

    1981-10-25

    A new manganese-containing acid phosphatase has been isolated and crystallized from sweet potato tubers. The pure enzyme contains one atom of manganese per Mr = 110,000 polypeptide and shows phosphatase activity toward various phosphate substrates. The pH optimum of the enzyme was 5.8 and the enzyme activity was inhibited by Cu2+, Zn2+, Hg2+, AsO43-, and MoO42-. This stable metalloenzyme is red-violet in color with an intense absorption band at 515 nm (epsilon - 2460). Our electronic, circular dichroism, and electron spin resonance findings strongly indicate that the Mn-valence state of the native enzyme is trivalent. When the Mn-enzyme is excited by the 5145 A line of Ar+ laser, prominent Raman lines at 1230, 1298, 1508, and 1620 cm-1 were detected. This Raman spectrum can probably be interpreted in terms of internal vibration of a coordinated tyrosine phenolate anion. The tryptophan-modified enzyme showed a positive Raman band at 370 cm-1, which is preferentially assigned to a Mn(III)-S streching mode. The modification of the Mn-enzyme by N-bromosuccinimide led to a large decrease in the fluorescence intensity of 335 nm which was dominated by its tryptophan residues within a considerable hydrophobic environment. The acid phosphatase activity was significantly decreased by the tryptophan modification. With respect to the active site donor sets, the Mn(III)-containing acid phosphatase is distinctly different from the Zn(II)-containing alkaline phosphatase. Of interest is also the appreciable similarity of some enzymatic and spectroscopic properties between the present enzyme and uteroferrin.

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

    PubMed

    Zaera, Francisco

    2005-01-01

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

  5. Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo

    PubMed Central

    Lim, Sung In; Hahn, Young S.; Kwon, Inchan

    2015-01-01

    Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL × h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety. PMID:25862515

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

    SciTech Connect

    1987-12-31

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

  7. Comparison of muscle activation using various hand positions during the push-up exercise.

    PubMed

    Cogley, Robert M; Archambault, Teasha A; Fibeger, Jon F; Koverman, Mandy M; Youdas, James W; Hollman, John H

    2005-08-01

    Popular fitness literature suggests that varied hand placements during push-ups may isolate different muscles. Scientific literature, however, offers scant evidence that varied hand placements elicit different muscle responses. This study examined whether different levels of electromyographic (EMG) activity in the pectoralis major and triceps brachii muscles are required to perform push-ups from each of 3 different hand positions: shoulder width base, wide base, and narrow base hand placements. Forty subjects, 11 men and 29 women, performed 1 repetition of each push-up. The EMG activity for subjects' dominant arm pectoralis major and triceps brachii was recorded using surface electrodes. The EMG activity was greater in both muscle groups during push-ups performed from the narrow base hand position compared with the wide base position (p < 0.05). This study suggests that, if a goal is to induce greater muscle activation during exercise, then push-ups should be performed with hands in a narrow base position compared with a wide base position.

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

    PubMed

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

    2009-05-06

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

  9. Role of Positional Hydrophobicity in the Leishmanicidal Activity of Magainin 2

    PubMed Central

    Guerrero, Esther; Saugar, José María; Matsuzaki, Katsumi; Rivas, Luis

    2004-01-01

    The emergence of membrane-active antimicrobial peptides as new alternatives against pathogens with multiantibiotic resistance requires the design of better analogues. Among the different physicochemical parameters involved in the optimization of linear antimicrobial peptides, positional hydrophobicity has recently been incorporated. This takes into consideration the concept of the topological distribution of hydrophobic residues throughout the sequence rather than the classical concept of hydrophobicity as a global parameter of the peptide, calculated as the summation of the individual hydrophobicities of the residues. In order to assess the contribution of this parameter to the leishmanicidal mechanisms of magainin 2 analogues, the activities of two of these analogues, MG-H1 (GIKKFLHIIWKFIKAFVGEIMNS) and MG-H2 (IIKKFLHSIWKFGKAFVGEIMNI), which have similar charges, amino acid compositions, and hydrophobicities but different positional hydrophobicities, against Leishmania donovani promastigotes were assayed (T. Tachi, R. F. Epand, R. M. Epand, and K. Matsuzaki, Biochemistry 41:10723-10731, 2002). The activities were compared with that of the parental peptide, F5W-magainin 2 (GIGKWLHSAKKFGKAFVGEIMNS). The three peptides were active at micromolar concentrations, in the order MG-H2 > MG-H1 > F5W-magainin 2. These activities differ from their hemolytic and bactericidal activities. The results demonstrate that positional hydrophobicity, which reflects the presence of short stretches of sequences rich in hydrophobic amino acids, plays an important role in the activities of leishmanicidal peptides. PMID:15273109

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

    PubMed

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

    2007-05-29

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

  11. Influence of hip position and gender on active hip internal and external rotation.

    PubMed

    Simoneau, G G; Hoenig, K J; Lepley, J E; Papanek, P E

    1998-09-01

    A general lack of descriptive details exists for measurements of hip rotation range of motion. This study was designed to establish the influence of gender and hip flexion position on active range of motion of the hip in external and internal rotation. Sixty (39 females and 21 males) healthy college-age (21.8 +/- 1.7 years) subjects were studied. Hip rotation of the dominant leg of each subject was measured in the prone (hip near 0 degree of flexion) and seated (hip near 90 degrees of flexion) positions using a standard goniometer. Data were analyzed using an analysis of variance model. Pearson's r statistics were used to determine the degree of association between measurements of hip rotation made seated vs. prone. A statistically significant difference (p < 0.05) was found between mean hip external rotation (ER) measured seated (36 +/- 7 degrees) and mean hip ER measured prone (45 +/- 10 degrees). Conversely, mean hip internal rotation (IR) measured seated (33 +/- 7 degrees) was not statistically different than mean hip IR measured prone (36 +/- 9 degrees). Females had statistically more active hip internal and external rotation than males (p < 0.05). A moderate degree of association existed between measurements of hip ER taken in the prone vs. seated position (r = 0.57, p < 0.05). For IR, the degree of association between the two measurement positions was slightly higher (r = 0.72, p < 0.05). Unlike the amount of active hip internal rotation which showed little difference between measurements made prone vs. seated, our data indicate that measurement position had a significant effect on the amount of active range of motion of the hip in ER. These findings are clinically significant for they stress the importance of documenting measurement position. They also stress the need for representative norms to be established for each hip position and gender.

  12. Cortical activity differs between position- and force-control knee extension tasks.

    PubMed

    Poortvliet, Peter C; Tucker, Kylie J; Finnigan, Simon; Scott, Dion; Sowman, Paul; Hodges, Paul W

    2015-12-01

    Neural control differs between position- and force-control tasks as evident from divergent effects of fatigue and pain. Unlike force-control tasks, position-control tasks focus on a postural goal to maintain a joint angle. Cortical involvement is suggested to be less during postural control, but whether this differs between position- and force-control paradigms remains unclear. Coherence estimates the functional communication between spatially distinct active regions within the cortex (cortico-cortical coherence; CCC) and between the cortex and muscles (corticomuscular coherence; CMC). We investigated whether cortical involvement differed between force-control and more posturally focused, position-control tasks. Seventeen adults performed position- and force-control knee extensor efforts at a submaximal load (10 % maximum voluntary contraction). Surface electromyography was recorded from the right knee extensor and flexor muscles and brain activity using electroencephalography (EEG). CCC and CMC in the beta (13-30 Hz) and gamma (30-45 Hz) frequency bands were calculated between combinations of intra- and inter-hemispheric pairs of electrodes, and between four EEG electrodes that approximated the left motor cortical area, and right knee extensor EMG, respectively. Differences in EEG power and muscle activity were also calculated. CCC was greater across distributed regions in the force-control task. Beta EEG power in the left hemisphere was higher for the position-control task. Although averaged CMC data differed between tasks, there was no task difference for individual CMC data. Muscle activity and force did not differ between tasks. The results demonstrate differential cortical contributions to control force- versus position-control tasks. This might contribute to differences in performance outcomes of these tasks that have been shown previously.

  13. p21-activated kinase 4 regulates mitotic spindle positioning and orientation.

    PubMed

    Bompard, Guillaume; Morin, Nathalie

    2012-01-01

    During mitosis, microtubules (MTs) are massively rearranged into three sets of highly dynamic MTs that are nucleated from the centrosomes to form the mitotic spindle. Tight regulation of spindle positioning in the dividing cell and chromosome alignment at the center of the metaphase spindle are required to ensure perfect chromosome segregation and to position the cytokinetic furrow that will specify the two daughter cells. Spindle positioning requires regulation of MT dynamics, involving depolymerase activities together with cortical and kinetochore-mediated pushing and pulling forces acting on astral MTs and kinetochore fibres. These forces rely on MT motor activities. Cortical pulling forces exerted on astral MTs depend upon dynein/dynactin complexes and are essential in both symmetric and asymmetric cell division. A well-established spindle positioning pathway regulating the cortical targeting of dynein/dynactin involves the conserved LGN (Leu-Gly-Asn repeat-enriched-protein) and NuMA (microtubule binding nuclear mitotic apparatus protein) complex. Spindle orientation is also regulated by integrin-mediated cell adhesion and actin retraction fibres that respond to mechanical stress and are influenced by the microenvironment of the dividing cell. Altering the capture of astral MTs or modulating pulling forces affects spindle position, which can impair cell division, differentiation and embryogenesis. In this general scheme, the activity of mitotic kinases such as Auroras and Plk1 (Polo-like kinase 1) is crucial. Recently, the p21-activated kinases (PAKs) emerged as novel important players in mitotic progression. In our recent article, we demonstrated that PAK4 regulates spindle positioning in symmetric cell division. In this commentary, and in light of recent published studies, we discuss how PAK4 could participate in the regulation of mechanisms involved in spindle positioning and orientation.

  14. Active-site mutants of beta-lactamase: use of an inactive double mutant to study requirements for catalysis.

    PubMed

    Dalbadie-McFarland, G; Neitzel, J J; Richards, J H

    1986-01-28

    We have studied the catalytic activity and some other properties of mutants of Escherichia coli plasmid-encoded RTEM beta-lactamase (EC 3.5.2.6) with all combinations of serine and threonine residues at the active-site positions 70 and 71. (All natural beta-lactamases have conserved serine-70 and threonine-71.) From the inactive double mutant Ser-70----Thr, Thr-71----Ser [Dalbadie-McFarland, G., Cohen, L. W., Riggs, A. D., Morin, C., Itakura, K., & Richards, J. H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6409-6413], an active revertant, Thr-71----Ser (i.e., residue 70 in the double mutant had changed from threonine to the serine conserved at position 70 in the wild-type enzyme), was isolated by an approach that allows identification of active revertants in the absence of a background of wild-type enzyme. This mutant (Thr-71----Ser) has about 15% of the catalytic activity of wild-type beta-lactamase. The other possible mutant involving serine and threonine residues at positions 70 and 71 (Ser-70----Thr) shows no catalytic activity. The primary nucleophiles of a serine or a cysteine residue [Sigal, I. S., Harwood, B. G., & Arentzen, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] at position 70 thus seem essential for enzymatic activity. Compared to wild-type enzyme, all three mutants show significantly reduced resistance to proteolysis; for the active revertant (Thr-71----Ser), we have also observed reduced thermal stability and reduced resistance to denaturation by urea.

  15. 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.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2014-09-05

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro(216)-Phe(228) loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors.

  18. Active nucleosome positioning beyond intrinsic biophysics is revealed by in vitro reconstitution.

    PubMed

    Korber, Philipp

    2012-04-01

    Genome-wide nucleosome maps revealed well-positioned nucleosomes as a major theme in eukaryotic genome organization. Promoter regions often show a conserved pattern with an NDR (nucleosome-depleted region) from which regular nucleosomal arrays emanate. Three mechanistic contributions to such NDR-array-organization and nucleosome positioning in general are discussed: DNA sequence, DNA binders and DNA-templated processes. Especially, intrinsic biophysics of DNA sequence preferences for nucleosome formation was prominently suggested to explain the majority of nucleosome positions ('genomic code for nucleosome positioning'). Nonetheless, non-histone factors that bind DNA with high or low specificity, such as transcription factors or remodelling enzymes respectively and processes such as replication, transcription and the so-called 'statistical positioning' may be involved too. Recently, these models were tested for yeast by genome-wide reconstitution. DNA sequence preferences as probed by SGD (salt gradient dialysis) reconstitution generated many NDRs, but only few individual nucleosomes, at their proper positions, and no arrays. Addition of a yeast extract and ATP led to dramatically more in vivo-like nucleosome positioning, including regular arrays for the first time. This improvement depended essentially on the extract and ATP but not on transcription or replication. Nucleosome occupancy and close spacing were maintained around promoters, even at lower histone density, arguing for active packing of nucleosomes against the 5' ends of genes rather than statistical positioning. A first extract fractionation identified a direct, specific, necessary, but not sufficient role for the RSC (remodels the structure of chromatin) remodelling enzyme. Collectively, nucleosome positioning in yeast is actively determined by factors beyond intrinsic biophysics, and in steady-state rather than at equilibrium.

  19. HIV Serosorting, Status Disclosure, and Strategic Positioning Among Highly Sexually Active Gay and Bisexual Men

    PubMed Central

    Grov, Christian; Rendina, H. Jonathon; Moody, Raymond L.; Ventuneac, Ana

    2015-01-01

    Abstract Researchers have identified harm reduction strategies that gay, bisexual, and other men who have sex with men (GBMSM) use to reduce HIV transmission—including serosorting, status disclosure, and strategic positioning. We report on patterns of these behaviors among 376 highly sexually active (i.e., 9+partners, <90 days) GBMSM: mean age of 37, 49.5% men of color, 87.8% gay identified, 57.5% college educated. We found evidence that many men engaged in serosorting, status disclosure, and strategic positioning; however, rates varied based on the participant's HIV status. HIV-positive and HIV-negative men both engaged in sex with men of similar status more often than they engaged in sex with men known to be a different HIV status (i.e., serosorting). However, HIV-negative men disclosed their HIV-status with about half of their partners, whereas HIV-positive participants disclosed with only about one-third. With regard to strategic positioning, HIV-positive participants were the receptive partner about half the time with their HIV-negative partners and with their HIV-positive partners. In contrast, strategic positioning was very common among HIV-negative participants—they rarely bottomed with HIV-positive partners, bottomed about one-third of the time with status-unknown partners, and 42% of the time (on average) with HIV-negative partners. Highly sexually active GBMSM are a critical population in which to both investigate HIV prevention strategies as well as develop effective intervention programs. Providers and clinicians might be well served to include a wide range of behavioral harm reduction strategies in addition to condom use and biomedical approaches to reduce onward HIV transmission. PMID:26348322

  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. All the catalytic active sites of MoS2 for hydrogen evolution

    SciTech Connect

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

    2016-11-29

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

  2. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes

    PubMed Central

    Gryaznova, Yuliya; Caydasi, Ayse Koca; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-01-01

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. DOI: http://dx.doi.org/10.7554/eLife.14029.001 PMID:27159239

  3. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes.

    PubMed

    Gryaznova, Yuliya; Koca Caydasi, Ayse; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-05-09

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control.

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

    PubMed

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

    2012-01-01

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

  5. Single active-site histidine in D-xylose isomerase from Streptomyces violaceoruber. Identification by chemical derivatization and peptide mapping.

    PubMed

    Vangrysperre, W; Ampe, C; Kersters-Hilderson, H; Tempst, P

    1989-10-01

    Group-specific chemical modifications of D-xylose isomerase from Streptomyces violaceruber indicated that complete loss of activity is fully correlated with the acylation of a single histidine. Active-site protection, by the ligand combination of xylitol plus Mg2+, completely blocked diethyl pyrocarbonate derivatization of this particular residue [Vangrysperre, Callens, Kersters-Hilderson & De Bruyne (1988) Biochem. J. 250, 153-160]. Differential peptide mapping between D-xylose isomerase, which has previously been treated with diethyl pyrocarbonate in the presence or absence of xylitol plus Mg2+, allowed specific isolation and sequencing of a peptide containing this active-site histidine. For this purpose we used two essentially new techniques: first, a highly reproducible peptide cleavage protocol for protease-resistant, carbethoxylated proteins with guanidinium hydrochloride as denaturing agent and subtilisin for proteolysis; and second, reverse-phase liquid chromatography with dual-wavelength detection at 214 and 238 nm, and calculation of absorbance ratios. It allowed us to locate the single active-site histidine at position 54 in the primary structure of Streptomyces violaceoruber D-xylose isomerase. The sequence around this residue is conserved in D-xylose isomerases from a diversity of micro-organisms, suggesting that this is a structurally and/or functionally essential part of the molecule.

  6. Mechanistic Insights into the Bifunctional Non-Heme Iron Oxygenase Carbapenem Synthase by Active Site Saturation Mutagenesis

    PubMed Central

    Phelan, Ryan M.; Townsend, Craig A.

    2013-01-01

    The carbapenem class of β-lactam antibiotics is known for its remarkable potency, antibacterial spectrum and resistance to β-lactamase-mediated inactivation. While the biosynthesis of structurally “complex” carbapenems, such as thienamycin, share initial biochemical steps with carbapenem-3-carboxylate (“simple” carbapenem), the requisite inversion at C5 and formation of the characteristic α,β-unsaturated carboxylate are different in origin between the two groups. Here we consider carbapenem synthase, a mechanistically distinct bifunctional non-heme iron α-ketoglutarate-dependent enzyme responsible for the terminal reactions, C5 epimerization and desaturation, in simple carbapenem production. Interestingly, this enzyme accepts two stereoisomeric substrates and transforms each to a common active antibiotic. Owing both to enzyme and product instability, resort to saturation mutagenesis of active site and selected second-sphere residues gave clearly differing profiles of CarC tolerance to structural modification. Guided by a crystal structure and the mutational data, in silico docking was used to suggest the positioning of each disastereomeric substrate in the active site. The two orientations relative to the reactive iron-oxo center are manifest in the two distinct reactions, C5-epimerization and C2/3-desaturation. These observations favor a two-step reaction scheme involving two complete oxidative cycles as opposed to a single catalytic cycle in which an active site tyrosine, Tyr67, after hydrogen donation to achieve bicyclic ring inversion, is further hypothesized to serve as a radical carrier. PMID:23611403

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

  8. Binding site of amiloride to urokinase plasminogen activator depends on species.

    PubMed

    Jankun, J; Skrzypczak-Jankun, E

    2001-10-01

    A novel drug candidate is checked on its potency on animal models before it can advance to human phase of the research. Usually negative results on animal phase disqualify it. Targeting specific enzymes by small chemicals raises the question about the appropriateness of this approach. As an example, the urokinase (uPA) is recognized as an important enzyme responsible for cancer metastasis and angiogenesis. It is therefore important to ask the question if a small chemical will inhibit uPA of different species with the same or different potency. Using DNA sequence and known structure of uPA we have modeled 3D structures of uPAs for several different species. By theoretical calculations we have determined most probable structure of amiloride/uPAs complexes. Catalytic triad (B57, B102, B195) and specificity pocket (B187-B197, B212-B229) are highly conserved in all cases, and are the regions responsible for proteolytic activity and recognition of the substrate. Significant differences were observed in a different region (loop B93-B101), that we identified as binding site of amiloride to the tissue plasminogen activator (tPA). Although tPA shares the same function of activating plasminogen and it is structurally similar to uPA. Amiloride is a specific inhibitor of uPA but does not inhibit tPA. Our study shows that predicted position of amiloride depends on species and in some cases was located, as expected, in the specificity pocket, but in the other cases close to the loop B93-B101. This location could weaken affinity of binding or prevent inhibition of uPA. Therefore, drug screening and elimination process based solely on animal study, without careful structural analysis, could lead to the elimination of potential drugs for humans.

  9. Incidence and risk of central nervous system metastases as site of first recurrence in patients with HER2-positive breast cancer treated with adjuvant trastuzumab

    PubMed Central

    Olson, E. M.; Abdel-Rasoul, M.; Maly, J.; Wu, C. S.; Lin, N. U.; Shapiro, C. L.

    2013-01-01

    Background Central nervous system (CNS) disease as the site of first relapse after exposure to adjuvant trastuzumab has been reported. We carried out comprehensive meta-analysis to determine the risk of CNS metastases as the first site of recurrence in patients with HER2-positive breast cancer who received adjuvant trastuzumab. Methods Eligible studies include randomized trials of adjuvant trastuzumab administered for 1 year to patients with HER2-positive breast cancer who reported CNS metastases as first site of disease recurrence. Statistical analyses were conducted to calculate the incidence, relative risk (RR), and 95% confidence intervals (CIs) using fixed-effects inverse variance and random-effects models. Results A total of 9020 patients were included. The incidence of CNS metastases as first site of disease recurrence in HER2-positive patients receiving adjuvant trastuzumab was 2.56% (95% CI 2.07% to 3.01%) compared with 1.94% (95% CI 1.54% to 2.38%) in HER2-positive patients who did not receive adjuvant trastuzumab. The RR of the CNS as first site of relapse in trastuzumab-treated patients was 1.35 (95% CI 1.02–1.78, P = 0.038) compared with control arms without trastuzumab therapy. The ratio of CNS metastases to total number of recurrence events was 16.94% (95% CI 10.85% to 24.07%) and 8.33% (95% CI 6.49% to 10.86%) for the trastuzumab-treated and control groups, respectively. No statistically significant differences were found based on trastuzumab schedule or median follow-up time. No evidence of publication bias was observed. Conclusions Adjuvant trastuzumab is associated with a significant increased risk of CNS metastases as the site of first recurrence in HER2-positive breast cancer patients. PMID:23463626

  10. migS, a cis-acting site that affects bipolar positioning of oriC on the Escherichia coli chromosome

    PubMed Central

    Yamaichi, Yoshiharu; Niki, Hironori

    2004-01-01

    During replication of the Escherichia coli chromosome, the replicated Ori domains migrate towards opposite cell poles, suggesting that a cis-acting site for bipolar migration is located in this region. To identify this cis-acting site, a series of mutants was constructed by splitting subchromosomes from the original chromosome. One mutant, containing a 720 kb subchromosome, was found to be defective in the bipolar positioning of oriC. The creation of deletion mutants allowed the identification of migS, a 25 bp sequence, as the cis-acting site for the bipolar positioning of oriC. When migS was located at the replication terminus, the chromosomal segment showed bipolar positioning. migS was able to rescue bipolar migration of plasmid DNA containing a mutation in the SopABC partitioning system. Interestingly, multiple copies of the migS sequence on a plasmid in trans inhibited the bipolar positioning of oriC. Taken together, these findings indicate that migS plays a crucial role in the bipolar positioning of oriC. In addition, real-time analysis of the dynamic morphological changes of nucleoids in wild-type and migS mutants suggests that bipolar positioning of the replicated oriC contributes to nucleoid organization. PMID:14685268

  11. The complexities of measuring access to parks and physical activity sites in New York City: a quantitative and qualitative approach

    PubMed Central

    Maroko, Andrew R; Maantay, Juliana A; Sohler, Nancy L; Grady, Kristen L; Arno, Peter S

    2009-01-01

    Background Proximity to parks and physical activity sites has been linked to an increase in active behaviors, and positive impacts on health outcomes such as lower rates of cardiovascular disease, diabetes, and obesity. Since populations with a low socio-economic status as well as racial and ethnic minorities tend to experience worse health outcomes in the USA, access to parks and physical activity sites may be an environmental justice issue. Geographic Information systems were used to conduct quantitative and qualitative analyses of park accessibility in New York City, which included kernel density estimation, ordinary least squares (global) regression, geographically weighted (local) regression, and longitudinal case studies, consisting of field work and archival research. Accessibility was measured by both density of park acreage and density of physical activity sites. Independent variables included percent non-Hispanic black, percent Hispanic, percent below poverty, percent of adults without high school diploma, percent with limited English-speaking ability, and population density. Results The ordinary least squares linear regression found weak relationships in both the park acreage density and the physical activity site density models (Ra2 = .11 and .23, respectively; AIC = 7162 and 3529, respectively). Geographically weighted regression, however, suggested spatial non-stationarity in both models, indicating disparities in accessibility that vary over space with respect to magnitude and directionality of the relationships (AIC = 2014 and -1241, respectively). The qualitative analysis supported the findings of the local regression, confirming that although there is a geographically inequitable distribution of park space and physical activity sites, it is not globally predicted by race, ethnicity, or socio-economic status. Conclusion The combination of quantitative and qualitative analyses demonstrated the complexity of the issues around racial and ethnic

  12. Evaluation development for a physical activity positive youth development program for girls.

    PubMed

    Ullrich-French, Sarah; Cole, Amy N; Montgomery, Anna K

    2016-04-01

    Girls on the Run (GOTR) is an after school program for girls in third through fifth grade which utilizes a physical activity based positive youth development curriculum that culminates with completing a 5K run. Unfortunately, there is little empirical data documenting GOTR participant changes that align with the curriculum and describe the evaluation process. Therefore, this study presents an evaluation of GOTR consisting of three main processes: curriculum content analysis and stakeholder focus groups (N=11) to identify key outcomes of the program; community-based participatory research to collaborate with program personnel to further identify important outcomes; and the design and pilot testing of an instrument (N=104) for assessing changes in the theoretically grounded outcomes over time. Findings demonstrated a positive collaborative process that led to important information to be used for an impact evaluation of Girls on the Run and for future evaluation development efforts for physical activity based positive youth development.

  13. Positive Activities: Qualitative Research with Parents. Solutions Research. Research Report. DCSF-RR142

    ERIC Educational Resources Information Center

    Department for Children, Schools and Families, 2009

    2009-01-01

    This research was commissioned by COI and DCSF to understand in depth, the barriers, motivators and messages for parents to encourage participation in positive activities for young people. Within this the research was designed to understand the level of influence of parents in whether a young person participates/what a young person might…

  14. Towards an Understanding of Flow and Other Positive Experience Phenomena within Outdoor and Adventurous Activities.

    ERIC Educational Resources Information Center

    Boniface, Margaret R.

    2000-01-01

    People involved in adventurous activities frequently experience positive phenomena termed peak experience, peak performance, and "flow." Characteristics of these phenomena are compared, along with factors influencing the ability to experience such peak moments. Csikszentmihalyi's flow models are examined with regard to perceived levels…

  15. Research on acupuncture points and cortical functional activation position in cats by infrared imaging detection

    NASA Astrophysics Data System (ADS)

    Chen, Shuwang; Sha, Zhanyou; Wang, Shuhai; Wen, Huanming

    2007-12-01

    The research of the brain cognition is mainly to find out the activation position in brain according to the stimulation at present in the world. The research regards the animals as the experimental objects and explores the stimulation response on the cerebral cortex of acupuncture. It provides a new method, which can detect the activation position on the creatural cerebral cortex directly by middle-far infrared imaging. According to the theory of local temperature situation, the difference of cortical temperature maybe associate with the excitement of cortical nerve cells, the metabolism of local tissue and the local hemal circulation. Direct naked detection of temperature variety on cerebral cortex is applied by middle and far infrared imaging technology. So the activation position is ascertained. The effect of stimulation response is superior to other indirect methods. After removing the skulls on the head, full of cerebral cortex of a cat are exposed. By observing the infrared images and measuring the temperatures of the visual cerebral cortex during the process of acupuncturing, the points are used to judge the activation position. The variety in the cortical functional sections is corresponding to the result of the acupuncture points in terms of infrared images and temperatures. According to experimental results, we know that the variety of a cortical functional section is corresponding to a special acupuncture point exactly.

  16. Assessing the Positive Influence of Music Activities in Community Development Programs

    ERIC Educational Resources Information Center

    Dillon, Steve

    2006-01-01

    This article describes a framework for assessing the positive influence of music activities in community development programs. It examines hybrid music, health and rich media approaches to creative case study with the purpose of developing more compelling evidence based advocacy that examines the claims of a causal link. This preliminary study…

  17. A Systematic Review of the Relationship between Socio-Economic Position and Physical Activity

    ERIC Educational Resources Information Center

    Gidlow, Christopher; Johnston, Lynne Halley; Crone, Diane; Ellis, Naomi; James, David

    2006-01-01

    Objective: The aim of the present review was to examine epidemiological evidence to determine if there is strong evidence of a positive gradient of increasing physical activity across the socio-economic strata, and how relationships are affected by socio-economic measurement. Design: Systematic review. Method: A search of major databases was…

  18. Physical Activity and Positive Youth Development: Impact of a School-Based Program

    ERIC Educational Resources Information Center

    Madsen, Kristine A.; Hicks, Katherine; Thompson, Hannah

    2011-01-01

    Background: Protective factors associated with positive youth development predict health and education outcomes. This study explored trends in these protective factors and in physical activity among low-income students, and determined the impact of a school-based youth development program on these trends. Methods: This study used a…

  19. Students' Use of Extra-Curricular Activities for Positional Advantage in Competitive Job Markets

    ERIC Educational Resources Information Center

    Roulin, Nicolas; Bangerter, Adrian

    2013-01-01

    With the rise of mass higher education, competition between graduates in the labour market is increasing. Students are aware that their degree will not guarantee them a job and realise they should add value and distinction to their credentials to achieve a positional advantage. Participation in extra-curricular activities (ECAs) is one such…

  20. 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

  1. The Influence of Artificially Introduced N-Glycosylation Sites on the In Vitro Activity of Xenopus laevis Erythropoietin

    PubMed Central

    Nagasawa, Kazumichi; Meguro, Mizue; Sato, Kei; Tanizaki, Yuta; Nogawa-Kosaka, Nami; Kato, Takashi

    2015-01-01

    Erythropoietin (EPO), the primary regulator of erythropoiesis, is a heavily glycosylated protein found in humans and several other mammals. Intriguingly, we have previously found that EPO in Xenopus laevis (xlEPO) has no N-glycosylation sites, and cross-reacts with the human EPO (huEPO) receptor despite low homology with huEPO. In this study, we introduced N-glycosylation sites into wild-type xlEPO at the positions homologous to those in huEPO, and tested whether the glycosylated mutein retained its biological activity. Seven xlEPO muteins, containing 1–3 additional N-linked carbohydrates at positions 24, 38, and/or 83, were expressed in COS-1 cells. The muteins exhibited lower secretion efficiency, higher hydrophilicity, and stronger acidic properties than the wild type. All muteins stimulated the proliferation of both cell lines, xlEPO receptor-expressing xlEPOR-FDC/P2 cells and huEPO receptor-expressing UT-7/EPO cells, in a dose-dependent manner. Thus, the muteins retained their in vitro biological activities. The maximum effect on xlEPOR-FDC/P2 proliferation was decreased by the addition of N-linked carbohydrates, but that on UT-7/EPO proliferation was not changed, indicating that the muteins act as partial agonists to the xlEPO receptor, and near-full agonists to the huEPO receptor. Hence, the EPO-EPOR binding site in X. laevis locates the distal region of artificially introduced three N-glycosylation sites, demonstrating that the vital conformation to exert biological activity is conserved between humans and X. laevis, despite the low similarity in primary structures of EPO and EPOR. PMID:25898205

  2. Neural substrates associated with evaluative processing during co-activation of positivity and negativity: a PET investigation.

    PubMed

    Jung, Young Chul; An, Suk Kyoon; Seok, Jeong Ho; Kim, Jae Seung; Oh, Seung Jun; Moon, Dae Hyuk; Kim, Jae-Jin

    2006-10-01

    Affective symmetries, such as the positivity offset and negativity bias, have been postulated to be attributable to distinct activation functions of the positive and negative affect systems. We investigated the neural substrates that are engaged when the positive and negative affect systems undergo parallel and integrative processing. Eleven subjects were scanned using H(2)(15)O PET during choosing the subjective feeling produced by a stimulation pair of pictures or words. Four different conditions were designed for contrast: pure positivity, pure negativity, positivity offset, and negativity bias. The dorsolateral prefrontal activation was associated with positivity offset and negativity bias condition, whereas the ventromedial prefrontal activation, together with limbic and subcortical activations, was associated with pure positivity and pure negativity condition. The results indicated that positivity offset and negativity bias are not merely due to asymmetric activations of the positive and negative systems, but integrative processing of higher neocortical levels is involved.

  3. Activity of the Aurora kinase inhibitor VX-680 against Bcr/Abl-positive acute lymphoblastic leukemias.

    PubMed

    Fei, Fei; Stoddart, Sonia; Groffen, John; Heisterkamp, Nora

    2010-05-01

    The emergence of resistance to tyrosine kinase inhibitors due to point mutations in Bcr/Abl is a challenging problem for Philadelphia chromosome-positive (Ph-positive) acute lymphoblastic leukemia (ALL) patients, especially for those with the T315I mutation, against which neither nilotinib or dasatinib shows significant activity. VX-680 is a pan-Aurora kinase inhibitor active against all Bcr/Abl proteins but has not been extensively examined in preclinical models of Ph-positive ALL. Here, we have tested VX-680 for the treatment of Bcr/Abl-positive ALL when leukemic cells are protected by the presence of stroma. Under these conditions, VX-680 showed significant effects on primary human Ph-positive ALL cells both with and without the T315I mutation, including ablation of tyrosine phosphorylation downstream of Bcr/Abl, decreased viability, and induction of apoptosis. However, drug treatment of human Ph-positive ALL cells for 3 days followed by drug removal allowed the outgrowth of abnormal cells 21 days later, and on culture of mouse Bcr/Abl ALL cells on stroma with lower concentrations of VX-680, drug-resistant cells emerged. Combined treatment of human ALL cells lacking the T315I mutation with both VX-680 and dasatinib caused significantly more cytotoxicity than each drug alone. We suggest that use of VX-680 together with a second effective drug as first-line treatment for Ph-positive ALL is likely to be safer and more useful than second-line treatment with VX-680 as monotherapy for drug-resistant T315I Ph-positive ALL.

  4. Di-substituted cyclohexyl derivatives bind to two identical sites with positive cooperativity on the voltage-gated potassium channel, K(v)1.3.

    PubMed

    Schmalhofer, William A; Slaughter, Robert S; Matyskiela, Mary; Felix, John P; Tang, Yui S; Rupprecht, Kathleen; Kaczorowski, Gregory J; Garcia, Maria L

    2003-04-29

    Di-substituted cyclohexyl (DSC) derivatives inhibit the voltage-gated potassium channel, K(v)1.3, and have immunosuppressant activity (Schmalhofer et al. (2002) Biochemistry 41, 7781-7794). This class of inhibitors displays Hill coefficients of near 2 in functional assays, and trans DSC analogues appear to selectively interact with K(v)1.3 channel conformations related to C-type inactivation. To further understand the details of the DSC inhibitor interaction with potassium channels, trans-1-(N-n-propylcarbamoyloxy)-4-phenyl-4-(3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl)cyclo-hexane (trans-NPCO-DSC) was radiolabeled with tritium, and its binding characteristics to K(v)1.3 channels were determined. Specific binding of [(3)H]-trans-NPCO-DSC to K(v)1.3 channels is a saturable, time-dependent, and fully reversible process. Saturation binding isotherms and competition binding experiments are consistent with the presence of two receptor sites for DSC derivatives on the K(v)1.3 channel that display positive allosteric cooperativity. The high affinity interaction of [(3)H]-trans-NPCO-DSC with K(v)1.3 channels appears to correlate with the rates of C-type inactivation of the channel. These data, taken together, mark the first demonstration of the existence of multiple binding sites for an inhibitor of an ion channel and suggest that the high affinity interaction of trans-NPCO-DSC and similar inhibitors with K(v)1.3 channels could be exploited for the development of selective molecules that target this protein.

  5. 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.

  6. Thermally Activated Site Exchange and Quantum Exchange Coupling Processes in Unsymmetrical Trihydride Osmium Compounds.

    PubMed

    Castillo, Amaya; Barea, Guada; Esteruelas, Miguel A.; Lahoz, Fernando J.; LLedós, Agustí; Maseras, Feliu; Modrego, Javier; Oñate, Enrique; Oro, Luis A.; Ruiz, Natividad; Sola, Eduardo

    1999-04-19

    Reaction of the hexahydride complex OsH(6)(P(i)Pr(3))(2) (1) with pyridine-2-thiol leads to the trihydride derivative OsH(3){kappa-N,kappa-S-(2-Spy)}(P(i)Pr(3))(2) (2). The structure of 2 has been determined by X-ray diffraction. The geometry around the osmium atom can be described as a distorted pentagonal bipyramid with the phosphine ligands occupying axial positions. The equatorial plane contains the pyridine-2-thiolato group, attached through a bite angle of 65.7(1) degrees, and the three hydride ligands. The theoretical structure determination of the model complex OsH(3){kappa-N,kappa-S-(2-Spy)}(PH(3))(2) (2a) reveals that the hydride ligands form a triangle with sides of 1.623, 1.714, and 2.873 Å, respectively. A topological analysis of the electron density of 2a indicates that there is no significant electron density connecting the hydrogen atoms of the OsH(3) unit. In solution, the hydride ligands of 2 undergo two different thermally activated site exchange processes, which involve the central hydride with each hydride ligand situated close to the donor atoms of the chelate group. The activation barriers of both processes are similar. Theoretical calculations suggest that the transition states have a cis-hydride-dihydrogen nature. In addition to the thermally activated exchange processes, complex 2 shows quantum exchange coupling between the central hydride and the one situated close to the sulfur atom of the pyridine-2-thiolato group. The reactions of 1 with L-valine and 2-hydroxypyridine afford OsH(3){kappa-N,kappa-O-OC(O)CH[CH(CH(3))(2)]NH(2)}(P(i)Pr(3))(2) (3) and OsH(3){kappa-N,kappa-O-(2-Opy)}(P(i)Pr(3))(2) (4) respectively, which according to their spectroscopic data have a similar structure to that of 2. In solution, the hydride ligands of 3 and 4 also undergo two different thermally activated site exchange processes. However, they do not show quantum exchange coupling. The tetranuclear complexes [(P(i)Pr(3))(2)H(3)Os(&mgr;-biim)M(TFB)](2) [M = Rh

  7. Arm position influences the activation patterns of trunk muscles during trunk range-of-motion movements.

    PubMed

    Siu, Aaron; Schinkel-Ivy, Alison; Drake, Janessa Dm

    2016-10-01

    To understand the activation patterns of the trunk musculature, it is also important to consider the implications of adjacent structures such as the upper limbs, and the muscles that act to move the arms. This study investigated the effects of arm positions on the activation patterns and co-activation of the trunk musculature and muscles that move the arm during trunk range-of-motion movements (maximum trunk axial twist, flexion, and lateral bend). Fifteen males and fifteen females, asymptomatic for low back pain, performed maximum trunk range-of-motion movements, with three arm positions for axial twist (loose, crossed, abducted) and two positions for flexion and lateral bend (loose, crossed). Electromyographical data were collected for eight muscles bilaterally, and activation signals were cross-correlated between trunk muscles and the muscles that move the arms (upper trapezius, latissimus dorsi). Results revealed consistently greater muscle co-activation (higher cross-correlation coefficients) between the trunk muscles and upper trapezius for the abducted arm position during maximum trunk axial twist, while results for the latissimus dorsi-trunk pairings were more dependent on the specific trunk muscles (either abdominal or back) and latissimus dorsi muscle (either right or left side), as well as the range-of-motion movement. The findings of this study contribute to the understanding of interactions between the upper limbs and trunk, and highlight the influence of arm positions on the trunk musculature. In addition, the comparison of the present results to those of individuals with back or shoulder conditions may ultimately aid in elucidating underlying mechanisms or contributing factors to those conditions.

  8. 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.

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

    PubMed

    Foo, Guo Shiou; Sievers, Carsten

    2015-02-01

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

  10. 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

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

    PubMed Central

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

    2016-01-01

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

  12. [Antimicrobial spectrum of dalbavancin. Mechanism of action and in vitro activity against Gram-positive microorganisms].

    PubMed

    Cercenado, Emilia

    2017-01-01

    Because of the increase in bacterial resistance, there is a need for new antimicrobial agents. Dalbavancin is a semisynthetic glycopeptide that inhibits the late stages of bacterial cell wall synthesis in the same way as vancomycin, but in addition, its lipophilic side chain anchors dalbavancin to the cellular membrane and allows enhanced activity compared with that of vancomycin. Dalbavancin possesses a broad spectrum of in vitro activity against Gram-positive aerobic and anaerobic microorganisms, being 4-8 times more potent than vancomycin. The spectrum of dalbavancin includes staphylococci, enterococci, streptococci, and anaerobic Gram-positive cocci and bacilli. It is active against different species of multiresistant microorganisms, including methicillin-resistant Staphylococcus aureus and penicillin-resistant viridans streptococci and Streptococcus pneumoniae. Although it shows in vitro activity against Enterococcus spp., it is inactive against isolates expressing the VanA phenotype of vancomycin resistance. It also shows slow bactericidal activity against S. aureus, coagulase-negative staphylococci, and Streptococcus pyogenes. In general, the MIC90 (minimum inhibitory concentration 90%) against the majority of the microorganisms is 0.06mg/L and, more than 98% of the isolates that have been tested are inhibited at concentrations of ≤ 0.12mg/L. Dalbavancin is an interesting addition to the therapeutic armamentarium for the treatment of infections caused by Gram-positive microorganisms, including multidrug-resistant isolates.

  13. Theoretical analysis of the relationship between positive/negative cooperativity and enzyme activation/inhibition.

    PubMed

    Ge, Hao; Qian, Min

    2009-09-01

    Cooperativity is one of the "paradigms" in enzyme kinetics and molecular biology. But the classical textbook treatment of enzyme kinetics always indeed separates the concepts of positive/negative cooperativity from enzyme activation/inhibition, at least partially. Few theoretical analysis of their relationship has been discussed, although its experimental investigations might date back at least to 1970s. In the present paper, we try to apply the change of free energy as a connective parameter for investigating the relationship between positive/negative cooperativity and enzyme activation/inhibition through several classic equilibrium binding models. It is explicitly shown that the terms of positive/negative cooperativity could be equivalently regarded as enzyme activation/inhibition of the saturation function induced by the substrate molecule itself rather than any other additional effectors. Moreover, both the degree of cooperativity phenomenon and the degree of enzyme activation/inhibition monotonically increase with the change of free energy. Note that this result is quite different from the idea of relating cooperativity to the concepts of "substrate activation/inhibition", which is identified when at high substrate concentrations the reaction rate decreases instead of tending towards the maximum velocity, since it always needs a second substrate molecule.

  14. Positive And Negative Feedback Loops Coupled By Common Transcription Activator And Repressor

    NASA Astrophysics Data System (ADS)

    Sielewiesiuk, Jan; Łopaciuk, Agata

    2015-03-01

    Dynamical systems consisting of two interlocked loops with negative and positive feedback have been studied using the linear analysis of stability and numerical solutions. Conditions for saddle-node bifurcation were formulated in a general form. Conditions for Hopf bifurcations were found in a few symmetrical cases. Auto-oscillations, when they exist, are generated by the negative feedback repressive loop. This loop determines the frequency and amplitude of oscillations. The positive feedback loop of activation slightly modifies the oscillations. Oscillations are possible when the difference between Hilll's coefficients of the repression and activation is sufficiently high. The highly cooperative activation loop with a fast turnover slows down or even makes the oscillations impossible. The system under consideration can constitute a component of epigenetic or enzymatic regulation network.

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

    SciTech Connect

    Spannaus, Ralf; Bodem, Jochen

    2014-04-15

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

  16. Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations

    PubMed Central

    Sun, Zhizeng; Mehta, Shrenik C.; Adamski, Carolyn J.; Gibbs, Richard A.; Palzkill, Timothy

    2016-01-01

    CphA is a Zn2+-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function. PMID:27616327

  17. Picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus

    PubMed Central

    Flather, Dylan; Semler, Bert L.

    2015-01-01

    The compartmentalization of DNA replication and gene transcription in the nucleus and protein production in the cytoplasm is a defining feature of eukaryotic cells. The nucleus functions to maintain the integrity of the nuclear genome of the cell and to control gene expression based on intracellular and environmental signals received through the cytoplasm. The spatial separation of the major processes that lead to the expression of protein-coding genes establishes the necessity of a transport network to allow biomolecules to translocate between these two regions of the cell. The nucleocytoplasmic transport network is therefore essential for regulating normal cellular functioning. The Picornaviridae virus family is one of many viral families that disrupt the nucleocytoplasmic trafficking of cells to promote viral replication. Picornaviruses contain positive-sense, single-stranded RNA genomes and replicate in the cytoplasm of infected cells. As a result of the limited coding capacity of these viruses, cellular proteins are required by these intracellular parasites for both translation and genomic RNA replication. Being of messenger RNA polarity, a picornavirus genome can immediately be translated upon entering the cell cytoplasm. However, the replication of viral RNA requires the activity of RNA-binding proteins, many of which function in host gene expression, and are consequently localized to the nucleus. As a result, picornaviruses disrupt nucleocytoplasmic trafficking to exploit protein functions normally localized to a different cellular compartment from which they translate their genome to facilitate efficient replication. Furthermore, picornavirus proteins are also known to enter the nucleus of infected cells to limit host-cell transcription and down-regulate innate antiviral responses. The interactions of picornavirus proteins and host-cell nuclei are extensive, required for a productive infection, and are the focus of this review. PMID:26150805

  18. Catalytic efficiency of HAP phytases is determined by a key residue in close proximity to the active site.

    PubMed

    Fu, Dawei; Li, Zhongyuan; Huang, Huoqing; Yuan, Tiezheng; Shi, Pengjun; Luo, Huiying; Meng, Kun; Yang, Peilong; Yao, Bin

    2011-05-01

    The maximum activity of Yersinia enterocolitica phytase (YeAPPA) occurs at pH 5.0 and 45 °C, and notably, its specific activity (3.28 ± 0.24 U mg(-1)) is 800-fold less than that of its Yersinia kristeensenii homolog (YkAPPA; 88% amino acid sequence identity). Sequence alignment and molecular modeling show that the arginine at position 79 (Arg79) in YeAPPA corresponding to Gly in YkAPPA as well as other histidine acid phosphatase (HAP) phytases is the only non-conserved residue near the catalytic site. To characterize the effects of the corresponding residue on the specific activities of HAP phytases, Escherichia coli EcAPPA, a well-characterized phytase with a known crystal structure, was selected for mutagenesis-its Gly73 was replaced with Arg, Asp, Glu, Ser, Thr, Leu, or Tyr. The results show that the specific activities of all of the corresponding EcAPPA mutants (17-2,400 U mg(-1)) were less than that of the wild-type phytase (3,524 U mg(-1)), and the activity levels were approximately proportional to the molecular volumes of the substituted residues' side chains. Site-directed replacement of Arg79 in YeAPPA (corresponding to Gly73 of EcAPPA) with Ser, Leu, and Gly largely increased the specific activity, which further verified the key role of the residue at position 79 for determining phytase activity. Thus, a new determinant that influences the catalytic efficiency of HAP phytases has been identified.

  19. 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.

  20. 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.

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

    PubMed Central

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

    2009-01-01

    Summary Uroporphyrinogen Decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of the four acetate side chains on the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer with the active site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single chain protein (scURO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposible with wild-type activity and have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of scURO-D resulted in approximately half of wild-type activity. The distribution of reaction intermediates was the same for mutant and wild-type sequences, and was unaltered in a competition experiment using the I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function, and suggest that the dimeric structure of URO-D is required to achieve conformational stability and create a large active site cleft. PMID:19362562

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

    SciTech Connect

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

    2009-01-01

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

  3. 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.

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

    PubMed Central

    Hanakahi, L. A.; Maizels, Nancy

    2000-01-01

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

  5. The divide within: Older active ICT users position themselves against different 'Others'.

    PubMed

    Kania-Lundholm, Magdalena; Torres, Sandra

    2015-12-01

    Although research into older people's internet usage patterns is rapidly growing, their understandings of digital technologies, particularly in relation to how these are informed by their understandings of aging and old age, remain unexplored. This is the case because research on older active ICT users tends to regard old age as an empirically interesting part of the life-course as opposed to a theoretically profuse source of information about why and how older people engage with digital technologies. This article explores - through focus group interviews with 30 older adults (aged 66-89) - the ways in which the social position of old age is used by older active ICT users in order to make sense of how and why they engage with these technologies. In this article, positioning theory is used to shed light on how the older people interviewed positioned themselves as 'active older users' in the interviews. The analysis brings to the fore the divide that older people themselves create as they discursively position themselves against different types of ICT users and non-users (young and old) when describing how and why they engage with digital technologies.

  6. Automated docking of {alpha}-(1,4)- and {alpha}-(1,6)-linked glucosyl trisaccharides in the glucoamylase active site

    SciTech Connect

    Countinho, P.M.; Reilly, P.J.; Dowd, M.K.

    1998-06-01

    Low-energy conformers of five {alpha}-(1,4)- and {alpha}-(1,6)-linked glucosyl trisaccharides were flexibly docked into the glucoamylase active site using AutoDock 2.2. To ensure that all significant conformational space was searched, the starting trisaccharide conformers for docking were all possible combinations of the corresponding disaccharide low-energy conformers. All docked trisaccharides occupied subsites {minus}1 and +1 in very similar modes to those of corresponding nonreducing-end disaccharides. For linear substrates, full binding at subsite +2 occurred only when the substrate reducing end was {alpha}-(1,4)-linked, with hydrogen-bonding with the hydroxy-methyl group being the only polar interaction there. Given the absence of other important interactions at this subsite, multiple substrate conformations are allowed. For the one docked branched substrate, steric hindrance in the {alpha}-(1,6)-glycosidic oxygen suggests that the active-site residues have to change position for hydrolysis to occur. Subsite +1 of the glucoamylase active site allows flexibility in binding but, at least in Aspergillus glucoamylases, subsite +2 selectively binds substrates {alpha}-(1,4)-linked between subsites +1 and +2. Enzyme engineering to limit substrate flexibility at subsite +2 could improve glucoamylase industrial properties.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  9. 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.

  10. SUPPLEMENTAL COLUMBIA RIVER PROTECTION ACTIVITIES AT THE DEPARTMENT OF ENERGY HANFORD SITE 2008 TECHNICAL REVIEW

    SciTech Connect

    Looney, B; Dawn S. Kaback, D; Eugene L. LeBoeuf, E; Joe Rossabi, J; Karen L. Skubal, K; David L. Cocke, D; Paul C. Deutsch, P

    2008-09-30

    Beginning in 2006, the US Department of Energy (DOE) supported nine applied research projects to improve the protection of the Columbia River and mitigate the impacts of Hanford Site groundwater. These projects were funded through a supplemental Congressional budget allocation, and are now in various stages of completion in accordance with the research plans. The DOE Office of Environmental Management Groundwater and Soil Cleanup Technologies (EM-22) sponsored a technical peer review meeting for these projects in Richland WA, July 28-31, 2008. The overall objective of the peer review is to provide information to support DOE decisions about the status and potential future application of the various technologies. The charge for the peer review panel was to develop recommendations for each of the nine 'technologies'. Team members for the July 2008 review were Brian Looney, Gene LeBoeuf, Dawn Kaback, Karen Skubal, Joe Rossabi, Paul Deutsch, and David Cocke. Previous project reviews were held in May 2007 and March-May of 2006. The team used the following four rating categories for projects: (a) Incorporate the technology/strategy in ongoing and future EM activities; (b) Finish existing scope of applied research and determine potential for EM activities when research program is finished; (c) Discontinue current development activities and do not incorporate technology/strategy into ongoing and future EM activities unless a significant and compelling change in potential viability is documented; and (d) Supplement original funded work to obtain the data needed to support a DOE decision to incorporate the technology into ongoing and future EM activities. The supplemental funding portfolio included two projects that addressed strontium, five projects that addressed chromium, one project that addressed uranium and one project that addressed carbon tetrachloride. The projects ranged from in situ treatment methods for immobilizing contaminants using chemical-based methods such

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

    DTIC Science & Technology

    1993-07-19

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

  12. Influence Of Scapular Position On The Core Musculature Activation In The Prone Plank Exercise.

    PubMed

    Cortell-Tormo, Juan M; García-Jaén, Miguel; Chulvi-Medrano, Iván; Hernández-Sánchez, Sergio; Lucas-Cuevas, Ángel G; Tortosa-Martínez, Juan

    2016-10-26

    Prone plank is a widely used exercise in core stability training. Research has shown that pelvic tilt plays an important role on the electromyographical (EMG) activation of core musculature. However, the influence of scapular position on EMG activation is currently unknown. Therefore, this study evaluated the influence of scapular position on the core muscles during a prone plank. Surface electromyography of the rectus abdominis (RA), external oblique (EO), internal oblique (IO) and erector spinae (ES) was collected in fifteen participants (10 men, 5 women). Four variations of the prone plank were evaluated: scapular abduction with anterior (ABANT) and posterior (ABRET) pelvic tilt; and scapular adduction with anterior (ADANT) and posterior (ADRET) pelvic tilt. Individual muscle EMG and overall EMG for each plank exercise was analyzed. Joint positions were controlled with a 2D kinematic analysis. Ratings of perceived effort (RPE) were also registered. ADRET resulted in higher overall EMG activity compared to ABANT (p=0.04) and ADANT (p=0.04). Moreover, ADRET resulted in greater EMG activity compared to ADANT, ABANT, and ABRET for EO (p=0.000; p=0.000; p=0.035), IO (p=0.000; p=0.000; p=0.005) and ES (p=0.019; p=0.001; p=0.014). Regarding RA, ADRET was significantly higher compared to ADANT (p=0.002) and ABANT (p=0.005). Finally, ADRET provoked a higher RPE compared to ABANT (p=0.000), ABRET (p=0.001) and ADANT (p=0.015). These findings demonstrate the influence of the scapular and pelvic position on the EMG response of the core muscle groups analyzed in this study, and highlight the greater contribution of these muscles to the postural stabilizing demands during posterior pelvic tilt positions, particularly when the scapulae are in adduction.

  13. Gaze and hand position effects on finger-movement-related human brain activation.

    PubMed

    Bédard, Patrick; Sanes, Jerome N

    2009-02-01

    Humans commonly use their hands to move and to interact with their environment by processing visual and proprioceptive information to determine the location of a goal-object and the initial hand position. It remains elusive, however, how the human brain fully uses this sensory information to generate accurate movements. In monkeys, it appears that frontal and parietal areas use and combine gaze and hand signals to generate movements, whereas in humans, prior work has separately assessed how the brain uses these two signals. Here we investigated whether and how the human brain integrates gaze orientation and hand position during simple visually triggered finger tapping. We hypothesized that parietal, frontal, and subcortical regions involved in movement production would also exhibit modulation of movement-related activation as a function of gaze and hand positions. We used functional MRI to measure brain activation while healthy young adults performed a visually cued finger movement and fixed gaze at each of three locations and held the arm in two different configurations. We found several areas that exhibited activation related to a mixture of these hand and gaze positions; these included the sensory-motor cortex, supramarginal gyrus, superior parietal lobule, superior frontal gyrus, anterior cingulate, and left cerebellum. We also found regions within the left insula, left cuneus, left midcingulate gyrus, left putamen, and right tempo-occipital junction with activation driven only by gaze orientation. Finally, clusters with hand position effects were found in the cerebellum bilaterally. Our results indicate that these areas integrate at least two signals to perform visual-motor actions and that these could be used to subserve sensory-motor transformations.

  14. Use of Cellular Decapping Activators by Positive-Strand RNA Viruses

    PubMed Central

    Jungfleisch, Jennifer; Blasco-Moreno, Bernat; Díez, Juana

    2016-01-01

    Positive-strand RNA viruses have evolved multiple strategies to not only circumvent the hostile decay machinery but to trick it into being a priceless collaborator supporting viral RNA translation and replication. In this review, we describe the versatile interaction of positive-strand RNA viruses and the 5′-3′ mRNA decay machinery with a focus on the viral subversion of decapping activators. This highly conserved viral trickery is exemplified with the plant Brome mosaic virus, the animal Flock house virus and the human hepatitis C virus. PMID:28009841

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

    PubMed

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

    2014-07-01

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

  16. Two active site divalent ions in the crystal structure of the hammerhead ribozyme bound to a transition state analogue

    DOE PAGES

    Mir, Aamir; Golden, Barbara L.

    2015-11-09

    The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analogue vanadate reveals significant rearrangements relative to the previously determined structures. The active site contracts, bringing G10.1 closer to the cleavage site and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to O6 of G12. This metal ion is well-placed to help tune the pKA of G12. Finally, on the basis of this crystalmore » structure as well as a wealth of biochemical studies, in this paper we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid.« less

  17. Two active site divalent ions in the crystal structure of the hammerhead ribozyme bound to a transition state analogue

    SciTech Connect

    Mir, Aamir; Golden, Barbara L.

    2015-11-09

    The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analogue vanadate reveals significant rearrangements relative to the previously determined structures. The active site contracts, bringing G10.1 closer to the cleavage site and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to O6 of G12. This metal ion is well-placed to help tune the pKA of G12. Finally, on the basis of this crystal structure as well as a wealth of biochemical studies, in this paper we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid.

  18. Mapping the Zap-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells.

    PubMed Central

    Paz, P E; Wang, S; Clarke, H; Lu, X; Stokoe, D; Abo, A

    2001-01-01

    T-cell-receptor (TCR)-mediated LAT (linker for activation of T cells) phosphorylation is critical for the membrane recruitment of signalling complexes required for T-cell activation. Although tyrosine phosphorylation of LAT is required for recruitment and activation of signalling proteins, the molecular mechanism associated with this event is unclear. In the present study we reconstituted the LAT signalling pathway by demonstrating that a direct tyrosine phosphorylation of LAT with activated protein-tyrosine kinase Zap70 is necessary and sufficient for the association and activation of signalling proteins. Zap-70 efficiently phosphorylates LAT on tyrosine residues at positions 226, 191, 171, 132 and 127. By substituting these tyrosine residues in LAT with phenylalanine and by utilizing phosphorylated peptides derived from these sites, we mapped the tyrosine residues in LAT required for the direct interaction and activation of Vav, p85/p110alpha and phospholipase Cgamma1 (PLCgamma1). Our results indicate that Tyr(226) and Tyr(191) are required for Vav binding, whereas Tyr(171) and Tyr(132) are necessary for association and activation of phosphoinositide 3-kinase activity and PLCgamma1 respectively. Furthermore, by expression of LAT mutants in LAT-deficient T cells, we demonstrate that Tyr(191) and Tyr(171) are required for T-cell activation and Tyr(132) is required for the activation of PLCgamma1 and Ras signalling pathways. PMID:11368773

  19. Brain activation related to combinations of gaze position, visual input, and goal-directed hand movements.

    PubMed

    Bédard, Patrick; Wu, Min; Sanes, Jerome N

    2011-06-01

    Humans reach to and acquire objects by transforming visual targets into action commands. How the brain integrates goals specified in a visual framework to signals into a suitable framework for an action plan requires clarification whether visual input, per se, interacts with gaze position to formulate action plans. To further evaluate brain control of visual-motor integration, we assessed brain activation, using functional magnetic resonance imaging. Humans performed goal-directed movements toward visible or remembered targets while fixating gaze left or right from center. We dissociated movement planning from performance using a delayed-response task and manipulated target visibility by its availability throughout the delay or blanking it 500 ms after onset. We found strong effects of gaze orientation on brain activation during planning and interactive effects of target visibility and gaze orientation on movement-related activation during performance in parietal and premotor cortices (PM), cerebellum, and basal ganglia, with more activation for rightward gaze at a visible target and no gaze modulation for movements directed toward remembered targets. These results demonstrate effects of gaze position on PM and movement-related processes and provide new information how visual signals interact with gaze position in transforming visual inputs into motor goals.

  20. An Analysis of Muscle Activities of Healthy Women during Pilates Exercises in a Prone Position.

    PubMed

    Kim, Bo-In; Jung, Ju-Hyeon; Shim, Jemyung; Kwon, Hae-Yeon; Kim, Haroo

    2014-01-01

    [Purpose] This study analyzed the activities of the back and hip muscles during Pilates exercises conducted in a prone position. [Subjects] The subjects were 18 healthy women volunteers who had practiced at a Pilates center for more than three months. [Methods] The subjects performed three Pilates exercises. To examine muscle activity during the exercises, 8-channel surface electromyography (Noraxon USA, Inc., Scottsdale, AZ) was used. The surface electrodes were attached to the bilateral latissimus dorsi muscle, multifidus muscle, gluteus maximus, and semitendinous muscle. Three Pilates back exercises were compared: (1) double leg kick (DLK), (2) swimming (SW), and (3) leg beat (LB). Electrical muscle activation was normalized to maximal voluntary isometric contraction. Repeated measures analysis of variance was performed to assess the differences in activation levels among the exercises. [Results] The activity of the multifidus muscle was significantly high for the SW (52.3±11.0, 50.9±9.8) and LB exercises(51.8±12.8, 48.3±13.9) and the activity of the semitendinosus muscle was higher for the LB exercise (49.2±8.7, 52.9±9.3) than for the DLK and SW exercises. [Conclusion] These results may provide basic material for when Pilates exercises are performed in a prone position and may be useful information on clinical Pilates for rehabilitation programs.

  1. Visualization of positive transcription elongation factor b (P-TEFb) activation in living cells.

    PubMed

    Fujinaga, Koh; Luo, Zeping; Schaufele, Fred; Peterlin, B Matija

    2015-01-16

    Regulation of transcription elongation by positive transcription elongation factor b (P-TEFb) plays a central role in determining the state of cell activation, proliferation, and differentiation. In cells, P-TEFb exists in active and inactive forms. Its release from the inactive 7SK small nuclear ribonucleoprotein complex is a critical step for P-TEFb to activate transcription elongation. However, no good method exists to analyze this P-TEFb equilibrium in living cells. Only inaccurate and labor-intensive cell-free biochemical assays are currently available. In this study, we present the first experimental system to monitor P-TEFb activation in living cells. We created a bimolecular fluorescence complementation assay to detect interactions between P-TEFb and its substrate, the C-terminal domain of RNA polymerase II. When cells were treated with suberoylanilide hydroxamic acid, which releases P-TEFb from the 7SK small nuclear ribonucleoprotein, they turned green. Other known P-TEFb-releasing agents, including histone deacetylase inhibitors, bromodomain and extraterminal bromodomain inhibitors, and protein kinase C agonists, also scored positive in this assay. Finally, we identified 5'-azacytidine as a new P-TEFb-releasing agent. This release of P-TEFb correlated directly with activation of human HIV and HEXIM1 transcription. Thus, our visualization of P-TEFb activation by fluorescent complementation assay could be used to find new P-TEFb-releasing agents, compare different classes of agents, and assess their efficacy singly and/or in combination.

  2. Computational analysis of negative and positive allosteric modulator binding and function in metabotropic glutamate receptor 5 (in)activation.

    PubMed

    Dalton, James A R; Gómez-Santacana, Xavier; Llebaria, Amadeu; Giraldo, Jesús

    2014-05-27

    Metabotropic glutamate receptors (mGluRs) are high-profile G-protein coupled receptors drug targets because of their involvement in several neurological disease states, and mGluR5 in particular is a subtype whose controlled allosteric modulation, both positive and negative, can potentially be useful for the treatment of schizophrenia and relief of chronic pain, respectively. Here we model mGluR5 with a collection of positive and negative allosteric modulators (PAMs and NAMs) in both active and inactive receptor states, in a manner that is consistent with experimental information, using a specialized protocol that includes homology to increase docking accuracy, and receptor relaxation to generate an individual induced fit with each allosteric modulator. Results implicate two residues in particular for NAM and PAM function: NAM interaction with W785 for receptor inactivation, and NAM/PAM H-bonding with S809 for receptor (in)activation. Models suggest the orientation of the H-bond between allosteric modulator and S809, controlled by PAM/NAM chemistry, influences the position of TM7, which in turn influences the shape of the allosteric site, and potentially the receptor state. NAM-bound and PAM-bound mGluR5 models also reveal that although PAMs and NAMs bind in the same pocket and share similar binding modes, they have distinct effects on the conformation of the receptor. Our models, together with the identification of a possible activation mechanism, may be useful in the rational design of new allosteric modulators for mGluR5.

  3. The hydrogen chemistry of the FeMo-co active site of nitrogenase.

    PubMed

    Dance, Ian

    2005-08-10

    The chemical mechanism by which nitrogenase enzymes catalyze the hydrogenation of N(2) (and other multiply bonded substrates) at the N(c)Fe(7)MoS(9)(homocitrate) active site (FeMo-co) is unknown, despite the accumulation of much data on enzyme reactivity and the influences of key amino acids surrounding FeMo-co. The mutual influences of H(2), substrates, and the inhibitor CO on reactivity are key experimental tests for postulated mechanisms. Fundamental to all aspects of mechanism is the accumulation of H atoms (from e(-) + H(+)) on FeMo-co, and the generation and influences of coordinated H(2). Here, I argue that the first introduction of H is via a water chain terminating at water 679 (PDB structure , Azotobacter vinelandii) to one of the mu(3)-S atoms (S3B) of FeMo-co. Next, using validated density functional calculations of a full chemical representation of FeMo-co and its connected residues (alpha-275(Cys), alpha-442(His)), I have characterized more than 80 possibilities for the coordination of up to three H atoms, and H(2), and H + H(2), on the S2A, Fe2, S2B, Fe6, S3B domain of FeMo-co, which is favored by recent targeted mutagenesis results. Included are calculated reaction profiles for movements of H atoms (between S and Fe, and between Fe and Fe), for the generation of Fe-H(2), for association and dissociation of Fe-H(2) at various reduction levels, and for H/H(2) exchange. This is new hydrogen chemistry on an unprecedented coordination frame, with some similarities to established hydrogen coordination chemistry, and with unexpected and unprecedented structures such as Fe(S)(3)(H(2))(2)(H) octahedral coordination. General principles for the hydrogen chemistry of FeMo-co include (1) the stereochemical mobility of H bound to mu(3)-S, (2) the differentiated endo- and exo- positions at Fe for coordination of H and/or H(2), and (3) coordinative allosteric influences in which structural and dynamic aspects of coordination at one Fe atom are affected by

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

    PubMed

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

    2016-09-01

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

  5. 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.

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

    PubMed

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

    2017-04-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    1980-06-01

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

  9. 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.

  10. Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase.

    PubMed

    Liao, Der-Ing; Zheng, Ya-Jun; Viitanen, Paul V; Jordan, Douglas B

    2002-02-12

    X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO(4)(2-), E-SO(4)(2-)-Mg(2+), E-SO(4)(2)(-)-Mn(2+), E-SO(4)(2)(-)-Mn(2+)-glycerol, and E-SO(4)(2)(-)-Zn(2+) complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 A, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg(2+) cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg(2+)-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme.

  11. Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone 4-phosphate synthase

    SciTech Connect

    Liao, D.-I.; Zheng, Y.-J.; Viitanen, P.V.; Jordan, D.B.

    2010-03-08

    X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO{sub 4}{sup 2-}, E-{sub 4}{sup 2-}-Mg{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}-glycerol, and E-SO{sub 4}{sup 2-}-Zn{sup 2+} complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 {angstrom}, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg{sup 2+} cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg{sup 2+}-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme.

  12. 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.

  13. Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

    NASA Astrophysics Data System (ADS)

    Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

    2015-04-01

    On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

  14. Individual Public Transportation Accessibility is Positively Associated with Self-Reported Active Commuting

    PubMed Central

    Djurhuus, Sune; Hansen, Henning Sten; Aadahl, Mette; Glümer, Charlotte

    2014-01-01

    Background: Active commuters have lower risk of chronic disease. Understanding which of the, to some extent, modifiable characteristics of public transportation that facilitate its use is thus important in a public health perspective. The aim of the study was to examine the association between individual public transportation accessibility and self-reported active commuting, and whether the associations varied with commute distance, age, and gender. Methods: Twenty-eight thousand nine hundred twenty-eight commuters in The Capital Region of Denmark reported self-reported time spent either walking or cycling to work or study each day and the distance to work or study. Data were obtained from the Danish National Health Survey collected in February to April 2010. Individual accessibility by public transportation was calculated using a multi-modal network in a GIS. Multilevel logistic regression was used to analyze the association between accessibility, expressed as access area, and being an active commuter. Results: Public transport accessibility area based on all stops within walking and cycling distance was positively associated with being an active commuter. Distance to work, age, and gender modified the associations. Residing within 10 km commute distance and in areas of high accessibility was associated with being an active commuter and meeting the recommendations of physical activity. For the respondents above 29 years, individual public transportation accessibility was positively associated with being an active commuter. Women having high accessibility had significantly higher odds of being an active commuter compared to having a low accessibility. For men, the associations were insignificant. Conclusion: This study extends the knowledge about the driving forces of using public transportation for commuting by examining the individual public transportation accessibility. Findings suggest that transportation accessibility supports active commuting and planning

  15. Bactericidal Activity and Mechanism of Photoirradiated Polyphenols against Gram-Positive and -Negative Bacteria.

    PubMed

    Nakamura, Keisuke; Ishiyama, Kirika; Sheng, Hong; Ikai, Hiroyo; Kanno, Taro; Niwano, Yoshimi

    2015-09-09

    The bactericidal effect of various types of photoirradiated polyphenols against Gram-positive and -negative bacteria was evaluated in relation to the mode of action. Gram-positive bacteria (Enterococcus faecalis, Staphylococcus aureus, and Streptococcus mutans) and Gram-negative bacteria (Aggregatibacter actinomycetemcomitans, Escherichia coli, and Pseudomonas aeruginosa) suspended in a 1 mg/mL polyphenol aqueous solution (caffeic acid, gallic acid, chlorogenic acid, epigallocatechin, epigallocatechin gallate, and proanthocyanidin) were exposed to LED light (wavelength, 400 nm; irradiance, 260 mW/cm(2)) for 5 or 10 min. Caffeic acid and chlorogenic acid exerted the highest bactericidal activity followed by gallic acid and proanthocyanidin against both Gram-positive and -negative bacteria. It was also demonstrated that the disinfection treatment induced oxidative damage of bacterial DNA, which suggests that polyphenols are incorporated into bacterial cells. The present study suggests that blue light irradiation of polyphenols could be a novel disinfection treatment.

  16. Passive damping to enhance active positioning of a prototype lithography platen

    SciTech Connect

    Segalman, D.J.; Kipp, R.L.; Gregory, D.L.

    1994-12-31

    A viscoelastic tuned-mass damper was used to suppress specific structural modes of a prototype lithography platen. The platen is magnetically levitated and it is repositioned and held in position by a closed-loop feedback control system. Important capabilities of the platen control system are precise positioning and rapid repositioning, which tend to require high frequency bandwidth. The high bandwidth excites structural vibration modes which are disruptive to the control system. The present work was to develop and demonstrate a means to suppress these modes using passive vibration damping techniques. The motivation is to increase the robustness of the platen positioning and control system by reducing unwanted modal accelerations excited by high control system bandwidth. Activities performed and discussed in this paper include the analytical design of viscoelastic tuned-mass dampers and the demonstration/testing of their effectiveness on the platen while levitated and controlled.

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

    NASA Astrophysics Data System (ADS)

    Snyder, Benjamin E. R.; Vanelderen, Pieter; Bols, Max L.; Hallaert, Simon D.; Böttger, Lars H.; Ungur, Liviu; Pierloot, Kristine; Schoonheydt, Robert A.; Sels, Bert F.; Solomon, Edward I.

    2016-08-01

    An efficient catalytic process for converting methane into methanol could have far-reaching economic implications. Iron-containing zeolites (microporous aluminosilicate minerals) are noteworthy in this regard, having an outstanding ability to hydroxylate methane rapidly at room temperature to form methanol. Reactivity occurs at an extra-lattice active site called α-Fe(II), which is activated by nitrous oxide to form the reactive intermediate α-O; however, despite nearly three decades of research, the nature of the active site and the factors determining its exceptional reactivity are unclear. The main difficulty is that the reactive species—α-Fe(II) and α-O—are challenging to probe spectroscopically: data from bulk techniques such as X-ray absorption spectroscopy and magnetic susceptibility are complicated by contributions from inactive ‘spectator’ iron. Here we show that a site-selective spectroscopic method regularly used in bioinorganic chemistry can overcome this problem. Magnetic circular dichroism reveals α-Fe(II) to be a mononuclear, high-spin, square planar Fe(II) site, while the reactive intermediate, α-O, is a mononuclear, high-spin Fe(IV)=O species, whose exceptional reactivity derives from a constrained coordination geometry enforced by the zeolite lattice. These findings illustrate the value of our approach to exploring active sites in heterogeneous systems. The results also suggest that using matrix constraints to activate metal sites for function—producing what is known in the context of metalloenzymes as an ‘entatic’ state—might be a useful way to tune the activity of heterogeneous catalysts.

  18. Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases

    PubMed Central

    Bellini, Dom; Horrell, Sam; Hutchin, Andrew; Phippen, Curtis W.; Strange, Richard W.; Cai, Yuming; Wagner, Armin; Webb, Jeremy S.; Tews, Ivo; Walsh, Martin A.

    2017-01-01

    The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation. PMID:28186120