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Sample records for active site pore

  1. A microporous Cu-MOF with optimized open metal sites and pore spaces for high gas storage and active chemical fixation of CO2.

    PubMed

    Gao, Chao-Ying; Tian, Hong-Rui; Ai, Jing; Li, Lei-Jiao; Dang, Song; Lan, Ya-Qian; Sun, Zhong-Ming

    2016-09-25

    A microporous Cu-MOF with optimized open metal sites and pore space was constructed based on a designed bent ligand; it exhibits high-capacity multiple gas storage under atmospheric pressure and efficient catalytic activity for chemical fixation of CO2 under mild conditions.

  2. A microporous Cu-MOF with optimized open metal sites and pore spaces for high gas storage and active chemical fixation of CO2.

    PubMed

    Gao, Chao-Ying; Tian, Hong-Rui; Ai, Jing; Li, Lei-Jiao; Dang, Song; Lan, Ya-Qian; Sun, Zhong-Ming

    2016-09-25

    A microporous Cu-MOF with optimized open metal sites and pore space was constructed based on a designed bent ligand; it exhibits high-capacity multiple gas storage under atmospheric pressure and efficient catalytic activity for chemical fixation of CO2 under mild conditions. PMID:27550833

  3. The ryanodine receptor pore blocker neomycin also inhibits channel activity via a previously undescribed high-affinity Ca(2+) binding site.

    PubMed

    Laver, Derek R; Hamada, Tomoyo; Fessenden, James D; Ikemoto, Noriaki

    2007-12-01

    In this study, we present evidence for the mechanism of neomycin inhibition of skeletal ryanodine receptors (RyRs). In single-channel recordings, neomycin produced monophasic inhibition of RyR open probability and biphasic inhibition of [(3)H]ryanodine binding. The half-maximal inhibitory concentration (IC(50)) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca(2+)], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca(2+) binding site that causes allosteric inhibition. This novel Ca(2+)/neomycin binding site had a neomycin affinity of 100 nM: and a Ca(2+) affinity of 35 nM,: which is 30-fold higher than that of the well-described cytoplasmic Ca(2+) activation site. Therefore, a new high-affinity class of Ca(2+) binding site(s) on the RyR exists that mediates neomycin inhibition. Neomycin plugging of the channel pore induced brief (1-2 ms) conductance substates at 30% of the fully open conductance, whereas allosteric inhibition caused complete channel closure with durations that depended on the neomycin concentration. We quantitatively account for these results using a dual inhibition model for neomycin that incorporates voltage-dependent pore plugging and Ca(2+)-dependent allosteric inhibition.

  4. Visualization of enzyme activities inside earthworm pores

    NASA Astrophysics Data System (ADS)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  5. Active genes at the nuclear pore complex.

    PubMed

    Taddei, Angela

    2007-06-01

    The nucleus is spatially and functionally organized and its architecture is now seen as a key contributor to genome functions. A central component of this architecture is the nuclear envelope, which is studded with nuclear pore complexes that serve as gateways for communication between the nucleoplasm and cytoplasm. Although the nuclear periphery has traditionally been described as a repressive compartment and repository for gene-poor chromosome regions, several recent studies in yeast have demonstrated that repressive and activating domains can both be positioned at the periphery of the nucleus. Moreover, association with the nuclear envelope favors the expression of particular genes, demonstrating that nuclear organization can play an active role in gene regulation. PMID:17467257

  6. Submicroscopic pores grafted using the residual sites produced by swift heavy ions

    NASA Astrophysics Data System (ADS)

    Mazzei, R.; Betz, N.; Bermúdez, G. García; Massa, G.; Smolko, E.

    2005-07-01

    To produce nuclear track membranes (NTM) with submicroscopic pores poly(vinylidene difluoride) (PVDF) foils were irradiated with Cl, Ag and Pb ions. Then they were chemically etched for different times and grafted with acrylic acid. The grafting yields were determined by weight measurements as a function of ion fluence, etching time and also analysed using Fourier transform infrared spectroscopy. Both measurements suggest that the acrylic acid was grafted on the pore wall of the NTM using the active sites left by the ion beam.

  7. Moving metal ions through ferritin-protein nanocages from three-fold pores to catalytic sites.

    PubMed

    Tosha, Takehiko; Ng, Ho-Leung; Bhattasali, Onita; Alber, Tom; Theil, Elizabeth C

    2010-10-20

    Ferritin nanocages synthesize ferric oxide minerals, containing hundreds to thousands of Fe(III) diferric oxo/hydroxo complexes, by reactions of Fe(II) ions with O(2) at multiple di-iron catalytic centers. Ferric-oxy multimers, tetramers, and/or larger mineral nuclei form during postcatalytic transit through the protein cage, and mineral accretion occurs in the central cavity. We determined how Fe(II) substrates can access catalytic sites using frog M ferritins, active and inactivated by ligand substitution, crystallized with 2.0 M Mg(II) ± 0.1 M Co(II) for Co(II)-selective sites. Co(II) inhibited Fe(II) oxidation. High-resolution (<1.5 Å) crystal structures show (1) a line of metal ions, 15 Å long, which penetrates the cage and defines ion channels and internal pores to the nanocavity that link external pores to the cage interior, (2) metal ions near negatively charged residues at the channel exits and along the inner cavity surface that model Fe(II) transit to active sites, and (3) alternate side-chain conformations, absent in ferritins with catalysis eliminated by amino acid substitution, which support current models of protein dynamics and explain changes in Fe-Fe distances observed during catalysis. The new structural data identify a ∼27-Å path Fe(II) ions can follow through ferritin entry channels between external pores and the central cavity and along the cavity surface to the active sites where mineral synthesis begins. This "bucket brigade" for Fe(II) ion access to the ferritin catalytic sites not only increases understanding of biological nanomineral synthesis but also reveals unexpected design principles for protein cage-based catalysts and nanomaterials.

  8. "Opening" the ferritin pore for iron release by mutation of conserved amino acids at interhelix and loop sites.

    PubMed

    Jin, W; Takagi, H; Pancorbo, B; Theil, E C

    2001-06-26

    Ferritin concentrates, stores, and detoxifies iron in most organisms. The iron is a solid, ferric oxide mineral (< or =4500 Fe) inside the protein shell. Eight pores are formed by subunit trimers of the 24 subunit protein. A role for the protein in controlling reduction and dissolution of the iron mineral was suggested in preliminary experiments [Takagi et al. (1998) J. Biol. Chem. 273, 18685-18688] with a proline/leucine substitution near the pore. Localized pore disorder in frog L134P crystals coincided with enhanced iron exit, triggered by reduction. In this report, nine additional substitutions of conserved amino acids near L134 were studied for effects on iron release. Alterations of a conserved hydrophobic pair, a conserved ion pair, and a loop at the ferritin pores all increased iron exit (3-30-fold). Protein assembly was unchanged, except for a slight decrease in volume (measured by gel filtration); ferroxidase activity was still in the millisecond range, but a small decrease indicates slight alteration of the channel from the pore to the oxidation site. The sensitivity of reductive iron exit rates to changes in conserved residues near the ferritin pores, associated with localized unfolding, suggests that the structure around the ferritin pores is a target for regulated protein unfolding and iron release.

  9. Pore structure of the activated coconut shell charcoal carbon

    NASA Astrophysics Data System (ADS)

    Budi, E.; Nasbey, H.; Yuniarti, B. D. P.; Nurmayatri, Y.; Fahdiana, J.; Budi, A. S.

    2014-09-01

    The development of activated carbon from coconut shell charcoal has been investigated by using physical method to determine the influence of activation parameters in term of temperature, argon gas pressure and time period on the pore structure of the activated carbon. The coconut shell charcoal was produced by pyrolisis process at temperature of about 75 - 150 °C for 6 hours. The charcoal was activated at various temperature (532, 700 and 868 °C), argon gas pressure (6.59, 15 and 23.4 kgf/cm2) and time period of (10, 60 and 120 minutes). The results showed that the pores size were reduced and distributed uniformly as the activation parameters are increased.

  10. Performance of coarse pore filtration activated sludge system.

    PubMed

    Alavi Moghaddam, M R; Satoh, H; Mino, T

    2002-01-01

    A coarse pore filter can be applied inside the aeration tank to facilitate the separation of sludge from liquid instead of sedimentation. This filter has pores, which are irregular in shape, and the pore size is bigger than those of MF. The objectives of the study were to maintain as much as MLSS in the activated sludge process with coarse pore filter and to investigate the performance under high MLSS condition. Small-scale reactor results so far show good quality of effluent specially after starting the sludge bulking in the system in terms of SS, TOC, DOC and turbidity. The average carbon removal for 62 days operation of this system was about 94% (based on effluent DOC) and 87% (based on effluent TOC). The average sludge yield in this system is about 0.44 kg MLSS/kg TOC which is about 0.24 kg MLSS/kg BOD. This amount is less than those of conventional activated sludge and trickling filter.

  11. Estimation of the pore pressure distribution from three dimensional groundwater flow model at mine sites in Korea

    NASA Astrophysics Data System (ADS)

    Kang, Sangsoo; Jang, Myounghwan; Kim, Gyoungman; Kim, Donghui; Kim, Daehoon; Baek, Hwanjo

    2016-04-01

    Mining activities continually change the groundwater flow and associated pore pressure distributions within the rockmass around the mine openings or the open-pit bench during the operational periods. As the pore pressure distributions may substantially affect the mechanical behaviour or stability of the rockmass, it is important to monitor the variation of pore pressure incurred by mining operation. The pore pressure distributions within the rockmass can be derived using a two- or three-dimensional finite element groundwater flow model, adopted to simulate the groundwater flow. While the groundwater inflow at mines has generally been dealt with respect to the working environment, detailed case studies on the distribution of pore water pressure related to the stability analysis of mine openings have been relatively rare in Korea. Recently, however, as the health and safety problems are emerged for sustainable mining practice, these issues are of the major concerns for the mining industries. This study aims to establish a three dimensional groundwater flow model to estimate the pore pressure distributions in order to employ as an input parameter for numerical codes such as the FLAC 3D. Also, the groundwater flow simulated can be used for de-watering design at a mine site. The MINEDW code, a groundwater flow model code specifically developed to simulate the complicated hydro-geologic conditions related to mining, has mainly been used in this study. Based on the data collected from field surveys and literature reviews, a conceptual model was established and sensitivity analysis was performed.

  12. Characterization of a Ligand Binding Site in the Human Transient Receptor Potential Ankyrin 1 Pore

    PubMed Central

    Klement, Göran; Eisele, Lina; Malinowsky, David; Nolting, Andreas; Svensson, Mats; Terp, Gitte; Weigelt, Dirk; Dabrowski, Michael

    2013-01-01

    The pharmacology and regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel activity is intricate due to the physiological function as an integrator of multiple chemical, mechanical, and temperature stimuli as well as differences in species pharmacology. In this study, we describe and compare the current inhibition efficacy of human TRPA1 on three different TRPA1 antagonists. We used a homology model of TRPA1 based on Kv1.2 to select pore vestibule residues available for interaction with ligands entering the vestibule. Site-directed mutation constructs were expressed in Xenopus oocytes and their functionality and pharmacology assessed to support and improve our homology model. Based on the functional pharmacology results we propose an antagonist-binding site in the vestibule of the TRPA1 ion channel. We use the results to describe the proposed intravestibular ligand-binding site in TRPA1 in detail. Based on the single site substitutions, we designed a human TRPA1 receptor by substituting several residues in the vestibule and adjacent regions from the rat receptor to address and explain observed species pharmacology differences. In parallel, the lack of effect on HC-030031 inhibition by the vestibule substitutions suggests that this molecule interacts with TRPA1 via a binding site not situated in the vestibule. PMID:23442958

  13. Characterization of a ligand binding site in the human transient receptor potential ankyrin 1 pore.

    PubMed

    Klement, Göran; Eisele, Lina; Malinowsky, David; Nolting, Andreas; Svensson, Mats; Terp, Gitte; Weigelt, Dirk; Dabrowski, Michael

    2013-02-19

    The pharmacology and regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel activity is intricate due to the physiological function as an integrator of multiple chemical, mechanical, and temperature stimuli as well as differences in species pharmacology. In this study, we describe and compare the current inhibition efficacy of human TRPA1 on three different TRPA1 antagonists. We used a homology model of TRPA1 based on Kv1.2 to select pore vestibule residues available for interaction with ligands entering the vestibule. Site-directed mutation constructs were expressed in Xenopus oocytes and their functionality and pharmacology assessed to support and improve our homology model. Based on the functional pharmacology results we propose an antagonist-binding site in the vestibule of the TRPA1 ion channel. We use the results to describe the proposed intravestibular ligand-binding site in TRPA1 in detail. Based on the single site substitutions, we designed a human TRPA1 receptor by substituting several residues in the vestibule and adjacent regions from the rat receptor to address and explain observed species pharmacology differences. In parallel, the lack of effect on HC-030031 inhibition by the vestibule substitutions suggests that this molecule interacts with TRPA1 via a binding site not situated in the vestibule.

  14. Characterization of pores in high pressure die cast aluminum using active thermography and computed tomography

    NASA Astrophysics Data System (ADS)

    Maierhofer, Christiane; Myrach, Philipp; Röllig, Mathias; Jonietz, Florian; Illerhaus, Bernhard; Meinel, Dietmar; Richter, Uwe; Miksche, Ronald

    2016-02-01

    Larger high pressure die castings (HPDC) and decreasing wall thicknesses are raising the issue of casting defects like pores in aluminum structures. Properties of components are often strongly influenced by inner porosity. As these products are being established more and more in lightweight construction (e.g. automotive and other transport areas), non-destructive testing methods, which can be applied fast and on-site, are required for quality assurance. In this contribution, the application of active thermography for the direct detection of larger pores is demonstrated. The analysis of limits and accuracy of the method are completed by numerical simulation and the method is validated using computed tomography.

  15. Characteristics of fluoride in pore-water at accidental hydrofluoric acid spillage site, Gumi, Korea

    NASA Astrophysics Data System (ADS)

    Kwon, E. H.; Lee, H. A.; Lee, J.; Kim, D.; Lee, S.; Yoon, H. O.

    2015-12-01

    A leakage accident of hydrofluoric acid (HF) occurred in Gumi, South Korea at Sep. 2012. The study site is located in the borderline between a large-scale industrial complex and a rural area. The HF plume was made immediately, and moved toward the rural area through air. After the accident, 212 ha of farm land were influenced and most of crops were withered. To recover the soil, CaO was applied after six months. Although several studies have done to estimate the extension and movement of HF plume in the air and to assess the impact on human health or plant after the incident, the long-term fate of fluoride (F) in the affected soils is not identified clearly. Thus, this study aimed to understand the behavior of F in the soil after HF releasing from accident site through chemical analysis and geochemical modeling. Within the radius of 1 km of accident site, 16 pore-water and soil samples were collected. The semi-quantitative soil composition (i.e., Al, Ca, Fe, K, Mg, Mn, Na, P, Si, Ti), total F, total P, OM contents in soil, and soil pH have already been measured, and pore-water compositions are also identified. From these experimental and modeling data, we could be evaluate if impact of accident exists until now, and also could be select and identify existing form of fluoride in soil and pore-water.

  16. Mechanistic insights into the first Lygus-active β-pore forming protein.

    PubMed

    Jerga, Agoston; Chen, Danqi; Zhang, Chunfen; Fu, Jinping; Kouadio, Jean-Louis K; Wang, Yanfei; Duff, Stephen M G; Howard, Jennifer E; Rydel, Timothy J; Evdokimov, Artem G; Ramaseshadri, Parthasarathy; Evans, Adam; Bolognesi, Renata; Park, Yoonseong; Haas, Jeffrey A

    2016-06-15

    The cotton pests Lygus hesperus and Lygus lineolaris can be controlled by expressing Cry51Aa2.834_16 in cotton. Insecticidal activity of pore-forming proteins is generally associated with damage to the midgut epithelium due to pores, and their biological specificity results from a set of key determinants including proteolytic activation and receptor binding. We conducted mechanistic studies to gain insight into how the first Lygus-active β-pore forming protein variant functions. Biophysical characterization revealed that the full-length Cry51Aa2.834_16 was a stable dimer in solution, and when exposed to Lygus saliva or to trypsin, the protein underwent proteolytic cleavage at the C-terminus of each of the subunits, resulting in dissociation of the dimer to two separate monomers. The monomer showed tight binding to a specific protein in Lygus brush border membranes, and also formed a membrane-associated oligomeric complex both in vitro and in vivo. Chemically cross-linking the β-hairpin to the Cry51Aa2.834_16 body rendered the protein inactive, but still competent to compete for binding sites with the native protein in vivo. Our study suggests that disassociation of the Cry51Aa2.834_16 dimer into monomeric units with unoccupied head-region and sterically unhindered β-hairpin is required for brush border membrane binding, oligomerization, and the subsequent steps leading to insect mortality. PMID:27001423

  17. Evaluation of Colloid Retention Site Dominance in Variably Saturated Porous Media: An All Pores Pore-Scale Analysis

    NASA Astrophysics Data System (ADS)

    Morales, Veronica; Perez-Reche, Francisco; Holzner, Markus; Kinzelbach, Wolfgang

    2016-04-01

    It is well accepted that colloid and nanoparticle transport processes in porous media differ substantially between water saturated and unsaturated conditions. Differences are frequently ascribed to particle immobilization by association with interfaces with the gas, as well as to restrictions of the liquid medium through which colloids are transported. Yet, the current understanding of the importance of particle retention at gas interfaces is based on observations of single pores or two-dimensional pore network representations, leaving open the question of their statistical significance when all pores in the medium are considered. In order to address this question, column experiments were performed using a model porous medium of glass beads through which Silver particles were transported for conditions of varying water content and water chemistry. X-ray microtomography was subsequently employed as a non-destructive imaging technique to obtain pore-scale information of the entire column regarding: i) the presence and distribution of the main locations where colloids can become retained (interfaces with the water-solid, air-water, air-solid, and air-water-solid, grain-grain contacts, and the bulk liquid), ii) deposition profiles of colloids along the column classified by the available retention location, and iii) channel widths of 3-dimensional pore-water network representations. The results presented provide a direct statistical evaluation on the significance of colloid retention by attachment to interfaces or by strainig at contact points where multiple interfaces meet.

  18. Microbial sulfur metabolism evidenced from pore fluid isotope geochemistry at Site U1385

    NASA Astrophysics Data System (ADS)

    Turchyn, Alexandra V.; Antler, Gilad; Byrne, David; Miller, Madeline; Hodell, David A.

    2016-06-01

    At Site U1385, drilled during IODP Expedition 339 off the coast of Portugal on the continental slope, high-resolution sulfate concentration measurements in the pore fluids display non-steady-state behavior. At this site there is a zone of sulfate reduction in the uppermost seven meters of sediment, followed by a 38-meter interval where sulfate concentrations do not change, and finally sulfate concentrations are depleted to zero between 45 and 55 meters below seafloor. Below the sulfate minimum zone, there is abundant methane, suggesting that the lower sulfate consumption zone is coupled to anaerobic methane oxidation. We analyze pore water samples from IODP Site U1385 for sulfur and oxygen isotope ratios of dissolved sulfate, as well as the sulfur isotope composition of sedimentary pyrite. The sulfur isotopes in pore fluid sulfate display similar non-steady-state behavior similar to that of the sulfate concentrations, increasing over the uppermost zone of sulfate reduction and again over the lower zone of sulfate-driven anaerobic methane oxidation. The oxygen isotopes in sulfate increase to the 'apparent equilibrium' value in the uppermost zone of sulfate reduction and do not change further. Our calculations support the idea that sulfite to sulfide reduction is the limiting step in microbial sulfate reduction, and that the isotope fractionation expressed in the residual pore water sulfate pool is inversely proportional to the net sulfate reduction rate. The sulfur isotope composition of pyrite acquires one value in the uppermost sediments, which may be overprinted by a second value in the deeper sediments, possibly due to iron release during anaerobic methane oxidation or iron diffusion from a higher zone of bacterial iron reduction. Our results have implications for modeling the sulfur isotope composition of the pyrite burial flux in the global biogeochemical sulfur cycle.

  19. Nuclear pore ion channel activity in live syncytial nuclei.

    PubMed

    Bustamante, Jose Omar

    2002-05-01

    Nuclear pore complexes (NPCs) are important nanochannels for the control of gene activity and expression. Most of our knowledge of NPC function has been derived from isolated nuclei and permeabilized cells in cell lysates/extracts. Since recent patch-clamp work has challenged the dogma that NPCs are freely permeable to small particles, a preparation of isolated living nuclei in their native liquid environment was sought and found: the syncytial nuclei in the water of the coconut Cocos nucifera. These nuclei have all properties of NPC-mediated macromolecular transport (MMT) and express foreign green fluorescent protein (GFP) plasmids. They display chromatin movement, are created by particle aggregation or by division, can grow by throwing filaments to catch material, etc. This study shows, for the first time, that living NPCs engaged in MMT do not transport physiological ions - a phenomenon that explains observations of nucleocytoplasmic ion gradients. Since coconuts are inexpensive (less than US$1/nut per litre), this robust preparation may contribute to our understanding of NPCs and cell nucleus and to the development of biotechnologies for the production of DNA, RNA and proteins.

  20. Pore Water Extraction Test Near 241-SX Tank Farm at the Hanford Site, Washington, USA

    SciTech Connect

    Eberlein, Susan J.; Parker, Danny L.; Tabor, Cynthia L.; Holm, Melissa J.

    2013-11-11

    A proof-of-principle test is underway near the Hanford Site 241-SX Tank Farm. The test will evaluate a potential remediation technology that will use tank farm-deployable equipment to remove contaminated pore water from vadose zone soils. The test system was designed and built to address the constraints of working within a tank farm. Due to radioactive soil contamination and limitations in drilling near tanks, small-diameter direct push drilling techniques applicable to tank farms are being utilized for well placement. To address space and weight limitations in working around tanks and obstacles within tank farms, the above ground portions of the test system have been constructed to allow deployment flexibility. The test system utilizes low vacuum over a sealed well screen to establish flow into an extraction well. Extracted pore water is collected in a well sump,and then pumped to the surface using a small-diameter bladder pump.If pore water extraction using this system can be successfully demonstrated, it may be possible to target local contamination in the vadose zone around underground storage tanks. It is anticipated that the results of this proof-of-principle test will support future decision making regarding interim and final actions for soil contamination within the tank farms.

  1. Communication: Activation energy of tension-induced pore formation in lipid membranes.

    PubMed

    Karal, Mohammad Abu Sayem; Yamazaki, Masahito

    2015-08-28

    Tension plays a vital role in pore formation in biomembranes, but the mechanism of pore formation remains unclear. We investigated the temperature dependence of the rate constant of constant tension (σ)-induced pore formation in giant unilamellar vesicles of lipid membranes using an experimental method we developed. By analyzing this result, we determined the activation energy (Ua) of tension-induced pore formation as a function of tension. A constant (U0) that does not depend on tension was found to contribute significantly to Ua. Analysis of the activation energy clearly indicated that the dependence of Ua on σ in the classical theory is correct, but that the classical theory of pore formation is not entirely correct due to the presence of U0. We can reasonably consider that U0 is a nucleation free energy to form a hydrophilic pre-pore from a hydrophobic pre-pore or a region with lower lateral lipid density. After obtaining U0, the evolution of a pre-pore follows a classical theory. Our data provide valuable information that help explain the mechanism of tension-induced pore formation in biomembranes and lipid membranes.

  2. Origin and transport of pore fluids in the Nankai accretionary prism inferred from chemical and isotopic compositions of pore water at cold seep sites off Kumano

    NASA Astrophysics Data System (ADS)

    Toki, Tomohiro; Higa, Ryosaku; Ijiri, Akira; Tsunogai, Urumu; Ashi, Juichiro

    2014-12-01

    We used push corers during manned submersible dives to obtain sediment samples of up to 30 cm from the subseafloor at the Oomine Ridge. The concentrations of B in pore water extracted from the sediment samples from cold seep sites were higher than could be explained by organic matter decomposition, suggesting that the seepage fluid at the site was influenced by B derived from smectite-illite alteration, which occurs between 50°C and 160°C. Although the negative δ18OH2O and δDH2O values of the pore fluids cannot be explained by freshwater derived from clay mineral dehydration (CMD), we considered the contribution of pore fluids in the shallow sediments of the accretionary prism, which showed negative δ18OH2O and δDH2O values according to the results obtained during Integrated Ocean Drilling Program (IODP) Expeditions 315 and 316. We calculated the mixing ratios based on a four-end-member mixing model including freshwater derived from CMD, pore fluids in the shallow (SPF) accretionary prism sediment, seawater (SW), and freshwater derived from methane hydrate (MH) dissociation. However, the Oomine seep fluids were unable to be explained without four end members, suggesting that deep-sourced fluids in the accretionary prism influenced the seeping fluids from this area. This finding presents the first evidence of deep-sourced fluids at cold seep sites in the Oomine Ridge, indicating that a megasplay fault is a potential pathway for the deep-sourced fluids.

  3. Application of low-voltage field-emission SEM to the study of internal pore structures of activated carbon

    SciTech Connect

    Liu, J.; Ornberg, R.L.

    1996-12-31

    Activated carbon has interesting and useful properties for industrial applications. It has been used extensively in purification, separation, chemical recovery and catalysis. To achieve a predictable performance of activated carbon materials, it is necessary to develop a comprehensive understanding of the pore structure including pore size, pore shape, and pore surface chemistry. Macropores (> 50 nm), mesopores (2-50 nm) and micropores (< 2 nm) generally coexist in activated carbon. It is thus desirable to synthesize activated carbon with controlled pore structures to optimize its performance. We previously reported the characterization of the surface pore structure of activated carbon by field emission SEM (FESEM) and the examination of the internal pore structure by HAADF/HRTEM techniques. However, both HAADF and HRTEM techniques give only limited information about the carbon pore structure. We report here some preliminary observation of the internal pore structure of activated carbon by high resolution low voltage FESEM technique.

  4. Review: movement of mRNA from transcription site to nuclear pores.

    PubMed

    Politz, J C; Pederson, T

    2000-04-01

    Pre-mRNA is transcribed primarily from genes located at the interface between chromatin domains and the interchromatin space. After partial or complete processing and complexing with nuclear proteins, the transcripts leave their site of synthesis and travel through the interchromatin space to the nuclear pores for export to the cytoplasm. It is unclear whether transcripts are tethered within the interchromatin space and move toward the nuclear pores using a metabolic energy-requiring, directed mechanism or, alternatively, move randomly by a diffusion-based process. We discuss here recent progress in understanding this step of gene expression, including our experiments tracking the movement of intranuclear poly(A) RNA in living cells. Our results and those of others are most consistent with a model in which newly synthesized mRNAs diffuse throughout the interchromatin space until they randomly encounter and are captured by the export machinery. Because the export machinery appears to preferentially bind transport-competent mRNAs (complexed with the correct complement of nuclear proteins), this diffusion-based model for intranuclear RNA movement potentially allows for a significant level of posttranscriptional control of gene expression.

  5. Computer simulation of adsorption of a Stockmayer molecule chlorodifluoromethane in activated carbon slit pores

    NASA Astrophysics Data System (ADS)

    Jin, Wenzheng; Wang, Wenchuan

    2001-06-01

    The adsorption recovery of HCFC-22 is an urgent task for environment protection. In this work we use the grand canonical Monte Carlo (GCMC) method to simulate the adsorption of HCFC-22 in terms of a slit-like activated carbon adsorbent. In our simulation the molecule of HCFC-22 is modeled by using the effective Stockmayer potential developed by this group. Heterogeneous activated sites with three different densities are imposed on the carbon walls. Three types of simulations are carried out: (1) The Gibbs ensemble MC method is used to test the Stockmayer potential parameters recommended here. (2) The Widom test particle method is used for determining the relationship of the chemical potential and the bulk phase pressure. (3) The GCMC method is used for adsorption simulations. Simulation results suggest that the optimum carbon slit pore is of width 1.75 nm and the activated site density is 0.8 sites/nm2, when the adsorption is conducted at ambient temperature and pressure, and the exhaustion pressure is 0.011 MPa. In this case, the maximum amount of HCFC-22 would be recovered.

  6. Extracellular signal-regulated kinase 2 (ERK2) phosphorylation sites and docking domain on the nuclear pore complex protein Tpr cooperatively regulate ERK2-Tpr interaction.

    PubMed

    Vomastek, Tomás; Iwanicki, Marcin P; Burack, W Richard; Tiwari, Divya; Kumar, Devanand; Parsons, J Thomas; Weber, Michael J; Nandicoori, Vinay Kumar

    2008-11-01

    Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work, we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction. ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation. Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2 in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.

  7. Allosteric coupling of the inner activation gate to the outer pore of a potassium channel.

    PubMed

    Peters, Christian J; Fedida, David; Accili, Eric A

    2013-10-23

    In potassium channels, functional coupling of the inner and outer pore gates may result from energetic interactions between residues and conformational rearrangements that occur along a structural path between them. Here, we show that conservative mutations of a residue near the inner activation gate of the Shaker potassium channel (I470) modify the rate of C-type inactivation at the outer pore, pointing to this residue as part of a pathway that couples inner gate opening to changes in outer pore structure and reduction of ion flow. Because they remain equally sensitive to rises in extracellular potassium, altered inactivation rates of the mutant channels are not secondary to modified binding of potassium to the outer pore. Conservative mutations of I470 also influence the interaction of the Shaker N-terminus with the inner gate, which separately affects the outer pore.

  8. Pore size distribution and supercritical hydrogen adsorption in activated carbon fibers.

    PubMed

    Purewal, J J; Kabbour, H; Vajo, J J; Ahn, C C; Fultz, B

    2009-05-20

    Pore size distributions (PSD) and supercritical H2 isotherms have been measured for two activated carbon fiber (ACF) samples. The surface area and the PSD both depend on the degree of activation to which the ACF has been exposed. The low-surface-area ACF has a narrow PSD centered at 0.5 nm, while the high-surface-area ACF has a broad distribution of pore widths between 0.5 and 2 nm. The H2 adsorption enthalpy in the zero-coverage limit depends on the relative abundance of the smallest pores relative to the larger pores. Measurements of the H2 isosteric adsorption enthalpy indicate the presence of energy heterogeneity in both ACF samples. Additional measurements on a microporous, coconut-derived activated carbon are presented for reference.

  9. Geochemistry of surface and pore water at USGS coring sites in wetlands of South Florida, 1994 and 1995

    USGS Publications Warehouse

    Orem, William H.; Lerch, Harry E.; Rawlik, Peter

    2002-01-01

    In this report, we present preliminary data on surface and pore water geochemistry from 22 sites in south Florida sampled during 1994 and 1995. These results are part of a larger study designed to evaluate the role of biogeochemical processes in sediments in the cycling of carbon, nitrogen, phosphorus, and sulfur in the south Florida ecosystem. The data are briefly discussed in regard to regional trends in the concentrations of chemical species, and general diagenetic processes in sediments. These results are part of a larger study designed to evaluate the role of biogeochemical processes in sediments in the cycling of carbon, nitrogen, phosphorus, and sulfur in the south Florida ecosystem. These elements play a crucial role in regulating organic sedimentation, nutrient dynamics, redox conditions, and the biogeochemistry of mercury in the threatened wetlands of south Florida. Pore water samples for chemical analyis were obtained using a piston corer/squeezer designed to avoid compression of the sediment and avoid oxidation and contamination of the pore water samples. Results show distinct regional trends in both surface water and pore water geochemistry. Most chemical species in surface and pore water show peak concentrations in Water Conservation Area 2A, with diminishing concentrations to the south and west into Water Conservation Area 3A, and Everglades National Park. The largest differences observed were for phosphate and sulfide, with concentrations in pore waters in Water Conservation Area 2A up to 500x higher than concentrations observed in freshwater marsh areas of Water Conservation Area 3A and Everglades National Park. Sites near the Hillsboro Canal in Water Conservation Area 2A are heavily contaminated with both phosphorus and sulfur. Pore water profiles for dissolved reactive phosphate suggest that recycling of phosphorus at these contaminated sites occurs primarily in the upper 20 cm of sediment. High levels of sulfide in pore water in Water

  10. Role of nitrogen in pore development in activated carbon prepared by potassium carbonate activation of lignin

    NASA Astrophysics Data System (ADS)

    Tsubouchi, Naoto; Nishio, Megumi; Mochizuki, Yuuki

    2016-05-01

    The present work focuses on the role of nitrogen in the development of pores in activated carbon produced from lignin by K2CO3 activation, employing a fixed bed reactor under a high-purity He stream at temperatures of 500-900 °C. The specific surface area and pore volume obtained by activation of lignin alone are 230 m2/g and 0.13 cm3/g at 800 °C, and 540 m2/g and 0.31 cm3/g at 900 °C, respectively. Activation of a mixture of lignin and urea provides a significant increase in the surface area and volume, respectively reaching 3300-3400 m2/g and 2.0-2.3 cm3/g after holding at 800-900 °C for 1 h. Heating a lignin/urea/K2CO3 mixture leads to a significant decrease in the yield of released N-containing gases compared to the results for urea alone and a lignin/urea mixture, and most of the nitrogen in the urea is retained in the solid phase. X-ray photoelectron spectroscopy and X-ray diffraction analyses clearly show that part of the remaining nitrogen is present in heterocyclic structures (for example, pyridinic and pyrrolic nitrogen), and the rest is contained as KOCN at ≤600 °C and as KCN at ≥700 °C, such that the latter two compounds can be almost completely removed by water washing. The fate of nitrogen during heating of lignin/urea/K2CO3 and role of nitrogen in pore development in activated carbon are discussed on the basis of the results mentioned above.

  11. Study of the protein complex, pore diameter, and pore-forming activity of the Borrelia burgdorferi P13 porin.

    PubMed

    Bárcena-Uribarri, Iván; Thein, Marcus; Barbot, Mariam; Sans-Serramitjana, Eulalia; Bonde, Mari; Mentele, Reinhard; Lottspeich, Friedrich; Bergström, Sven; Benz, Roland

    2014-07-01

    P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 m KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to ±100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi.

  12. Adsorption of phenolics on activated carbon--impact of pore size and molecular oxygen.

    PubMed

    Lu, Qiuli; Sorial, George A

    2004-05-01

    The impact of pore size of activated carbon fibers (ACFs) on adsorption capacity and on the potential for oligomerization of phenolic compounds on the surface of ACFs in the presence of molecular oxygen has been investigated in this study. Compared with granular activated carbon (GAC), ACFs have unique pore size distributions, suitable to be used to elucidate the effect of pore structure on adsorption. Adsorption isotherm data were collected for o-cresol and 2-ethylphenol on four ACFs (ACC-10, ACC-15, ACC-20, and ACC-25) with different micropore volumes and BET surface area and on one type of GAC bituminous base. These isotherms were collected under anoxic (absence of molecular oxygen) and oxic (presence of molecular oxygen) conditions. No significant impact of the presence of molecular oxygen on adsorption capacity was noted for ACC-10. ACC-10 has an average pore width of 19.2 A and total pore volume of 0.43 cm3g(-1). On the other hand, for the remaining ACFs, which have larger average pore width and larger pore volume, significant increase in the adsorptive capacity had been observed when molecular oxygen was present. The GAC gave the greatest difference between anoxic and oxic conditions when compared to all the ACFs studied. Binary adsorption of o-cresol and 2-ethylphenol on ACFs with the least pore size (ACC-10) also showed no significant differences between oxic and anoxic environment. The binary system under both anoxic and oxic conditions was well predicted by the ideal adsorbed solution theory (IAST).

  13. The effects of pore structure on the behavior of water in lignite coal and activated carbon.

    PubMed

    Nwaka, Daniel; Tahmasebi, Arash; Tian, Lu; Yu, Jianglong

    2016-09-01

    The effects of physical structure (pore structure) on behavior of water in lignite coal and activated carbon (AC) samples were investigated by using Differential Scanning Calorimetry (DSC) and low-temperature X-ray diffraction (XRD) techniques. AC samples with different pore structures were prepared at 800°C in steam and the results were compared with that of parent lignite coal. The DSC results confirmed the presence of two types of freezable water that freeze at -8°C (free water) and -42°C (freezable bound water). A shift in peak position of free water (FW) towards lower temperature was observed in AC samples compared to the lignite coal with decreasing water loading. The amount of free water (FW) increased with increasing gasification conversion. The amounts of free and freezable bound water (FBW) in AC samples were calculated and correlated to pore volume and average pore size. The amount of FW in AC samples is well correlated to the pore volume and average pore size of the samples, while an opposite trend was observed for FBW. The low-temperature XRD analysis confirmed the existence of non-freezable water (NFW) in coal and AC with the boundary between the freezable and non-freezable water (NFW) determined. PMID:27254256

  14. Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels

    PubMed Central

    Shaya, David; Findeisen, Felix; Abderemane-Ali, Fayal; Arrigoni, Cristina; Wong, Stephanie; Nurva, Shailika Reddy; Loussouarn, Gildas; Minor, Daniel L.

    2013-01-01

    Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail ‘neck’, are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the ‘outer ion’ site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies shows that this site forms a previously unknown determinant of CaV high affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily. PMID:24120938

  15. Influence of pore size distribution on the adsorption of phenol on PET-based activated carbons.

    PubMed

    Lorenc-Grabowska, Ewa; Diez, María A; Gryglewicz, Grazyna

    2016-05-01

    The role of pore size distribution in the adsorption of phenol in aqueous solutions on polyethylene terephthalate (PET)-based activated carbons (ACs) has been analyzed. The ACs were prepared from PET and mixtures of PET with coal-tar pitch (CTP) by means of carbonization and subsequent steam and carbon dioxide activation at 850 and 950 °C, respectively. The resultant ACs were characterized on the basis of similarities in their surface chemical features and differences in their micropore size distributions. The adsorption of phenol was carried out in static conditions at ambient temperature. The pseudo-second order kinetic model and Langmuir model were found to fit the experimental data very well. The different adsorption capacities of the ACs towards phenol were attributed to differences in their micropore size distributions. Adsorption capacity was favoured by the volume of pores with a size smaller than 1.4 nm; but restricted by pores smaller than 0.8 nm. PMID:26890386

  16. The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine.

    PubMed

    Syeda, Ruhma; Santos, Jose S; Montal, Mauricio

    2016-02-01

    KCNQ (voltage-gated K(+) channel family 7 (Kv7)) channels control cellular excitability and underlie the K(+) current sensitive to muscarinic receptor signaling (the M current) in sympathetic neurons. Here we show that the novel anti-epileptic drug retigabine (RTG) modulates channel function of pore-only modules (PMs) of the human Kv7.2 and Kv7.3 homomeric channels and of Kv7.2/3 heteromeric channels by prolonging the residence time in the open state. In addition, the Kv7 channel PMs are shown to recapitulate the single-channel permeation and pharmacological specificity characteristics of the corresponding full-length proteins in their native cellular context. A mutation (W265L) in the reconstituted Kv7.3 PM renders the channel insensitive to RTG and favors the conductive conformation of the PM, in agreement to what is observed when the Kv7.3 mutant is heterologously expressed. On the basis of the new findings and homology models of the closed and open conformations of the Kv7.3 PM, we propose a structural mechanism for the gating of the Kv7.3 PM and for the site of action of RTG as a Kv7.2/Kv7.3 K(+) current activator. The results validate the modular design of human Kv channels and highlight the PM as a high-fidelity target for drug screening of Kv channels.

  17. Breathing Stimulant Compounds Inhibit TASK-3 Potassium Channel Function Likely by Binding at a Common Site in the Channel Pore

    PubMed Central

    Chokshi, Rikki H.; Larsen, Aaron T.; Bhayana, Brijesh

    2015-01-01

    Compounds PKTHPP (1-{1-[6-(biphenyl-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]-pyrimidin-4-yl]piperidin-4-yl}propan-1-one), A1899 (2ʹ′-[(4-methoxybenzoylamino)methyl]biphenyl-2-carboxylic acid 2,4-difluorobenzylamide), and doxapram inhibit TASK-1 (KCNK3) and TASK-3 (KCNK9) tandem pore (K2P) potassium channel function and stimulate breathing. To better understand the molecular mechanism(s) of action of these drugs, we undertook studies to identify amino acid residues in the TASK-3 protein that mediate this inhibition. Guided by homology modeling and molecular docking, we hypothesized that PKTHPP and A1899 bind in the TASK-3 intracellular pore. To test our hypothesis, we mutated each residue in or near the predicted PKTHPP and A1899 binding site (residues 118–128 and 228–248), individually, to a negatively charged aspartate. We quantified each mutation's effect on TASK-3 potassium channel concentration response to PKTHPP. Studies were conducted on TASK-3 transiently expressed in Fischer rat thyroid epithelial monolayers; channel function was measured in an Ussing chamber. TASK-3 pore mutations at residues 122 (L122D, E, or K) and 236 (G236D) caused the IC50 of PKTHPP to increase more than 1000-fold. TASK-3 mutants L122D, G236D, L239D, and V242D were resistant to block by PKTHPP, A1899, and doxapram. Our data are consistent with a model in which breathing stimulant compounds PKTHPP, A1899, and doxapram inhibit TASK-3 function by binding at a common site within the channel intracellular pore region, although binding outside the channel pore cannot yet be excluded. PMID:26268529

  18. Capsules with highly active pores and interiors: versatile platforms at the nanoscale.

    PubMed

    Müller, Achim; Gouzerh, Pierre

    2014-04-22

    Spherical porous capsules offer new exciting approaches in chemistry, materials sciences, and in context of physical and biological phenomena. The underlying concepts are reported with particular emphasis on metal oxide based capsules of the {M132 } Keplerate type which display-due to their exceptional structural features and easy variation/derivatization as well as exchange of building units-an unmatched range of properties and offer unique opportunities for investigating a variety of basic aspects of nanoscience, including the discovery of some new phenomena, especially those related to hydrophobicity issues that are of significance for everyday life. This relies in particular on the existence of a large number of flexible crown ether type pores/channels and the possibility of changing the interior from completely hydrophilic to completely hydrophobic due to the presence of numerous easily exchangeable internal ligands/functionalities; the capsules can even be constructed so that they enclose a large number of highly active Lewis and Brønsted acid sites. The manifold of possible applications/uses are outlined as subtitles with reference to results as well as possible future studies. There are, among many others, options to control passing cations under different internal frames allowing also their separations, to conduct studies about hydrophobic recognitions and clustering of biological interest in water, controlled internal ion transport, nanoscale dewetting, and to carry out basic as well as new types of reactions under confined conditions.

  19. Adsorption of hydrogen sulfide onto activated carbon fibers: effect of pore structure and surface chemistry.

    PubMed

    Feng, Wenguo; Kwon, Seokjoon; Borguet, Eric; Vidic, Radisav

    2005-12-15

    To understand the nature of H2S adsorption onto carbon surfaces under dry and anoxic conditions, the effects of carbon pore structure and surface chemistry were studied using activated carbon fibers (ACFs) with different pore structures and surface areas. Surface pretreatments, including oxidation and heattreatment, were conducted before adsorption/desorption tests in a fixed-bed reactor. Raw ACFs with higher surface area showed greater adsorption and retention of sulfur, and heat treatment further enhanced adsorption and retention of sulfur. The retained amount of hydrogen sulfide correlated well with the amount of basic functional groups on the carbon surface, while the desorbed amount reflected the effect of pore structure. Temperature-programmed desorption (TPD) and thermal gravimetric analysis (TGA) showed that the retained sulfurous compounds were strongly bonded to the carbon surface. In addition, surface chemistry of the sorbent might determine the predominant form of adsorbate on the surface. PMID:16475362

  20. Adsorption of hydrogen sulfide onto activated carbon fibers: effect of pore structure and surface chemistry.

    PubMed

    Feng, Wenguo; Kwon, Seokjoon; Borguet, Eric; Vidic, Radisav

    2005-12-15

    To understand the nature of H2S adsorption onto carbon surfaces under dry and anoxic conditions, the effects of carbon pore structure and surface chemistry were studied using activated carbon fibers (ACFs) with different pore structures and surface areas. Surface pretreatments, including oxidation and heattreatment, were conducted before adsorption/desorption tests in a fixed-bed reactor. Raw ACFs with higher surface area showed greater adsorption and retention of sulfur, and heat treatment further enhanced adsorption and retention of sulfur. The retained amount of hydrogen sulfide correlated well with the amount of basic functional groups on the carbon surface, while the desorbed amount reflected the effect of pore structure. Temperature-programmed desorption (TPD) and thermal gravimetric analysis (TGA) showed that the retained sulfurous compounds were strongly bonded to the carbon surface. In addition, surface chemistry of the sorbent might determine the predominant form of adsorbate on the surface.

  1. Quantification of active mitochondrial permeability transition pores using GNX-4975 inhibitor titrations provides insights into molecular identity

    PubMed Central

    Richardson, Andrew P.; Halestrap, Andrew P.

    2016-01-01

    Inhibition of the mitochondrial permeability transition pore (MPTP) by the novel inhibitor GNX-4975 was characterized. Titration of MPTP activity in de-energized rat liver mitochondria allowed determination of the number of GNX-4975-binding sites and their dissociation constant (Ki). Binding sites increased in number when MPTP opening was activated by increasing [Ca2+], phenylarsine oxide (PAO) or KSCN, and decreased when MPTP opening was inhibited with bongkrekic acid (BKA) or ADP. Values ranged between 9 and 50 pmol/mg of mitochondrial protein, but the Ki remained unchanged at ∼1.8 nM when the inhibitor was added before Ca2+. However, when GNX-4975 was added after Ca2+ it was much less potent with a Ki of ∼140 nM. These data imply that a protein conformational change is required to form the MPTP complex and generate the GNX-4975-binding site. Occupation of the latter with GNX-4975 prevents the Ca2+ binding that triggers pore opening. We also demonstrated that GNX-4975 stabilizes an interaction between the adenine nucleotide translocase (ANT), held in its ‘c’ conformation with carboxyatractyloside (CAT), and the phosphate carrier (PiC) bound to immobilized PAO. No components of the F1Fo-ATP synthase bound significantly to immobilized PAO. Our data are consistent with our previous proposal that the MPTP may form at an interface between the PiC and ANT (or other similar mitochondrial carrier proteins) when they adopt novel conformations induced by factors that sensitize the MPTP to [Ca2+]. We propose that GNX-4975 binds to this interface preventing a calcium-triggered event that opens the interface into a pore. PMID:26920024

  2. Quantification of active mitochondrial permeability transition pores using GNX-4975 inhibitor titrations provides insights into molecular identity.

    PubMed

    Richardson, Andrew P; Halestrap, Andrew P

    2016-05-01

    Inhibition of the mitochondrial permeability transition pore (MPTP) by the novel inhibitor GNX-4975 was characterized. Titration of MPTP activity in de-energized rat liver mitochondria allowed determination of the number of GNX-4975-binding sites and their dissociation constant (Ki). Binding sites increased in number when MPTP opening was activated by increasing [Ca(2+)], phenylarsine oxide (PAO) or KSCN, and decreased when MPTP opening was inhibited with bongkrekic acid (BKA) or ADP. Values ranged between 9 and 50 pmol/mg of mitochondrial protein, but the Ki remained unchanged at ∼1.8 nM when the inhibitor was added before Ca(2+) However, when GNX-4975 was added after Ca(2+) it was much less potent with a Ki of ∼140 nM. These data imply that a protein conformational change is required to form the MPTP complex and generate the GNX-4975-binding site. Occupation of the latter with GNX-4975 prevents the Ca(2+) binding that triggers pore opening. We also demonstrated that GNX-4975 stabilizes an interaction between the adenine nucleotide translocase (ANT), held in its 'c' conformation with carboxyatractyloside (CAT), and the phosphate carrier (PiC) bound to immobilized PAO. No components of the F1Fo-ATP synthase bound significantly to immobilized PAO. Our data are consistent with our previous proposal that the MPTP may form at an interface between the PiC and ANT (or other similar mitochondrial carrier proteins) when they adopt novel conformations induced by factors that sensitize the MPTP to [Ca(2+)]. We propose that GNX-4975 binds to this interface preventing a calcium-triggered event that opens the interface into a pore.

  3. Pore size assessment during corneal endothelial cells permeabilization by femtosecond laser activated carbon nanoparticles

    NASA Astrophysics Data System (ADS)

    Jumelle, C.; Mauclair, C.; Houzet, J.; Bernard, A.; He, Z.; Piselli, S.; Perrache, C.; Egaud, G.; Baubeau, E.; Gain, P.; Thuret, G.

    2015-07-01

    Corneal therapeutic molecules delivery represents a promising solution to maintain human corneal endothelial cells (HCECs) viability, but the difficulty is transport across cell membrane. A new delivery method published recently consists in ephemerally permeabilizing cell membranes using a photo-acoustic reaction produced by carbon nanoparticles (CNPs) and femtosecond laser (FsL). The aim of this work is to investigate the size of pores formed at cell membrane by this technique. To induce cell permeabilization, HCECs were put in contact with CNPs and irradiated with a 500 μm diameter Ti:Sa FsL focalized spot. Four sizes of marker molecules were delivered into HCECs to investigate pore sizes: calcein (1.2 nm), FITC-Dextran 4kDa (2.8 nm) and FITC-Dextran 70kDa (12 nm) and FITC-Dextran 2MDa (50 nm). Delivery of each molecule was assessed by flow cytometry, a technique able to measure their presence into cells. We showed that the delivery rate was dependent of their size. Calcein was delivered in 56.1±8.2% of HCECs, FITC-Dextran 4kDa in 42.2±3.5%, FITC-Dextran 70 kDa in 21.5±2.7% and finally FITC-Dextran 2MDa in 12.9±2.0%. It means that a large number of pores in the size ranging from 1.2 to 2.8 nm were formed. However, 12 nm and larger pores were almost half more infrequent. Pore sizes formed at cell membrane by the technique of cell permeabilization by FsL activated CNPs was investigated. The results indicated that the pore sizes are large enough for the efficient delivery of small, medium and big therapeutics molecules on HCECs by this technique.

  4. Pore-fluid chemistry along the main axis of an active lobe at the Congo deep-sea fan

    NASA Astrophysics Data System (ADS)

    Croguennec, C.; Ruffine, L.; Guyader, V.; Le Bruchec, J.; Ruesch, B.; Caprais, J.; Cathalot, C.; de Prunelé, A.; Germain, Y.; Bollinger, C.; Dennielou, B.; Olu, K.; Rabouille, C.

    2013-12-01

    The distal lobes of the Congo deep-sea fan constitute a unique in situ laboratory to study early diagenesis of marine sediments. They are located at water depth of about 5000 m and result from the deposition of sediment transported by turbidity currents along the channel-levee systems and submarine canyon connected to the Congo River. Thus, a huge amount of organic matter, transported from the river to the lobes, undergoes decomposition processes involving different oxidants present within the sedimentary column. This drastically changes the chemistry of the pore fluids, allowing the occurence of a succession of biogeochemical processes. The present study is part of an ongoing project which aims at better understanding the role and the fate of organic matter transported to the lobe systems, as well as its implication in the distribution of the living communities encountered there. Thus, pore fluids have been sampled from 8 Calypso cores in order to determine the concentration of dissolved elements. Five sites have been investigated: four of them are located along the main axis of a currently active lobe, the last one being located on a lobe disconnected from the chenals. The analyses of methane, major (Cl, SO4, Mg, Ca, K, Na) and minor (Sr, Ba, B, Li, Mn) elements have been carried out along with total alkalinity determination. The resulting profiles show a highly heterogeneous pore-fluid chemistry. Sulphate concentration near the seawater/sediment interface varies from 3 to 29 mM, indicating intense sulphate reduction. Surprisingly the lowest values are found at the site which is disconnected from the active lobe. The manganese cycle is well defined for all cores. The core recovered at the more distal lobe exhibits very peculiar pore-fluid profiles which are likely related to a geological event, most likely sediment slide and remobilization. References: Babonneau, N., Savoye, B., Cremer, M. & Klein, B., 2002. Morphology and architecture of the present canyon and

  5. X-ray microtomography shows pore structure and tortuosity in alkali-activated binders

    SciTech Connect

    Provis, John L.; Myers, Rupert J.; White, Claire E.; Rose, Volker; Deventer, Jannie S.J. van

    2012-06-15

    Durability of alkali-activated binders is of vital importance in their commercial application, and depends strongly on microstructure and pore network characteristics. X-ray microtomography ({mu}CT) offers, for the first time, direct insight into microstructural and pore structure characteristics in three dimensions. Here, {mu}CT is performed on a set of sodium metasilicate-activated fly ash/slag blends, using a synchrotron beamline instrument. Segmentation of the samples into pore and solid regions is then conducted, and pore tortuosity is calculated by a random walker method. Segmented porosity and diffusion tortuosity are correlated, and vary as a function of slag content (slag addition reduces porosity and increases tortuosity), and sample age (extended curing gives lower porosity and higher tortuosity). This is particularly notable for samples with {>=} 50% slag content, where a space-filling calcium (alumino)silicate hydrate gel provides porosity reductions which are not observed for the sodium aluminosilicate ('geopolymer') gels which do not chemically bind water of hydration.

  6. A systematic investigation of SO2 removal dynamics by coal-based activated cokes: The synergic enhancement effect of hierarchical pore configuration and gas components

    NASA Astrophysics Data System (ADS)

    Sun, Fei; Gao, Jihui; Liu, Xin; Tang, Xiaofan; Wu, Shaohua

    2015-12-01

    For the aim to break through the long-term roadblock to porous carbon based SO2 removal technology, typical coal-based activated cokes differing in terms of surface area, pore configuration and surface functional properties, were employed to investigate the SO2 removal dynamics. Among the employed activated cokes, the one with a hierarchically porous structure greatly enhanced the SO2 removal dynamics under the simulated flue gas compositions. More detailedly, SO2 separate adsorption property under normal temperature and pressure evidenced that monolayer SO2 molecules anchoring on micropore surface is the main adsorption pattern. The catalytic oxidation of SO2 follows the Eley-Rideal mechanism by which SO2 was firstly oxidized by molecular oxygen into SO3 which could depart partially to release the active sites for further adsorption. For the role of hierarchical pore configuration, it was proposed that micropores serve as gas adsorption and reaction accommodation, meso-/macropores act as byproduct H2SO4 transport and buffing reservoirs, which may in turn gives rise to the recovery of active sites in micropores and guarantees the continuous proceeding of sulfur-containing species transformation in the micropores. The present results suggest that pore configuration or interconnecting pattern, but not mere surface area or pore volume, should be favourably considered for optimizing heterogeneous gas-solid adsorption and reaction.

  7. Pre-activation of ice-nucleating particles by the pore condensation and freezing mechanism

    NASA Astrophysics Data System (ADS)

    Wagner, Robert; Kiselev, Alexei; Möhler, Ottmar; Saathoff, Harald; Steinke, Isabelle

    2016-02-01

    In spite of the resurgence in ice nucleation research a comparatively small number of studies deal with the phenomenon of pre-activation in heterogeneous ice nucleation. Fifty years ago, it was shown that various mineral dust and volcanic ash particles can be pre-activated to become nuclei for ice crystal formation even at temperatures as high as 270-271 K. Pre-activation was achieved under ice-subsaturated conditions without any preceding macroscopic ice growth by just temporarily cooling the particles to temperatures below 228 K. A two-step mechanism involving capillary condensation of supercooled water and subsequent homogeneous freezing was proposed to account for the particles' enhanced ice nucleation ability at high temperatures. This work reinvestigates the efficiency of the proposed pre-activation mechanism in temperature-cycling experiments performed in a large cloud chamber with suspended particles. We find the efficiency to be highest for the clay mineral illite as well as for highly porous materials like zeolite and diatomaceous earth, whereas most aerosols generated from desert dust surface samples did not reveal a measurable pre-activation ability. The pre-activation efficiency is linked to particle pores in a certain size range. As estimated by model calculations, only pores with diameters between about 5 and 8 nm contribute to pre-activation under ice-subsaturated conditions. This range is set by a combination of requirements from the negative Kelvin effect for condensation and a critical size of ice embryos for ice nucleation and melting. In contrast to the early study, pre-activation is only observed for temperatures below 260 K. Above that threshold, the particles' improved ice nucleation ability disappears due to the melting of ice in the pores.

  8. Piezo proteins are pore-forming subunits of mechanically activated channels.

    PubMed

    Coste, Bertrand; Xiao, Bailong; Santos, Jose S; Syeda, Ruhma; Grandl, Jörg; Spencer, Kathryn S; Kim, Sung Eun; Schmidt, Manuela; Mathur, Jayanti; Dubin, Adrienne E; Montal, Mauricio; Patapoutian, Ardem

    2012-02-19

    Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ∼1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.

  9. Salt site performance assessment activities

    SciTech Connect

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

    1983-01-01

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

  10. Dissociation of membrane binding and lytic activities of the lymphocyte pore-forming protein (perforin).

    PubMed

    Young, J D; Damiano, A; DiNome, M A; Leong, L G; Cohn, Z A

    1987-05-01

    Granules isolated from CTL and NK cells contain a cytolytic pore-forming protein (PFP/perforin). At low temperatures (on ice), PFP binds to erythrocyte membranes without producing hemolysis. Hemolysis occurs when the PFP-bound erythrocytes are warmed up to 37 degrees C, which defines a temperature-dependent, lytic (pore-formation) step distinct from the membrane-binding event. Ca2+ and neutral pH are required for both membrane binding and pore formation by PFP. Serum, LDL, HDL, and heparin inhibit the hemolytic activity of PFP by blocking its binding to lipid membranes. Lysis by PFP that has bound to erythrocyte membranes is no longer susceptible to the effect of these inhibitors. The hemolytic activities associated with intact granules and solubilized PFP show different requirements for Ca2+ and pH, indicating that cytolysis produced by isolated granules may involve an additional step, possibly fusion of granules with membranes. It is suggested that three distinct Ca2+- and pH-dependent events may be involved during cell killing by CTL and NK cells: fusion of cytoplasmic granules of effector cells with their plasma membrane, releasing PFP from cells; binding of the released PFP to target membranes; and insertion of monomers and the subsequent formation of lytic pores in the target membrane. The serum-mediated inhibition of membrane binding by PFP could prevent the accidental injury of bystander cells by cell-released PFP, but would allow cytolysis to proceed to completion once PFP has bound to the target membrane. PMID:3494808

  11. Pore-water extraction from unsaturated tuff by triaxial and one-dimensional compression methods, Nevada Test Site, Nevada

    SciTech Connect

    Mower, T.E.; Higgins, J.D.; Yang, In C.; Peters, C.A.

    1994-07-01

    The hydrologic system in the unsaturated tuff at Yucca Mountain, Nevada, is being evaluated for the US Department of Energy by the Yucca Mountain Project Branch of the US Geological Survey as a potential site for a high-level radioactive-waste repository. Part of this investigation includes a hydrochemical study that is being made to assess characteristics of the hydrologic system such as: traveltime, direction of flow, recharge and source relations, and types and magnitudes of chemical reactions in the unsaturated tuff. In addition, this hydrochemical information will be used in the study of the dispersive and corrosive effects of unsaturated-zone water on the radioactive-waste storage canisters. This report describes the design and validation of laboratory experimental procedures for extracting representative samples of uncontaminated pore water from welded and nonwelded, unsaturated tuffs from the Nevada Test Site.

  12. Pore-pressure sensitivities to dynamic strains: observations in active tectonic regions

    USGS Publications Warehouse

    Barbour, Andrew

    2015-01-01

    Triggered seismicity arising from dynamic stresses is often explained by the Mohr-Coulomb failure criterion, where elevated pore pressures reduce the effective strength of faults in fluid-saturated rock. The seismic response of a fluid-rock system naturally depends on its hydro-mechanical properties, but accurately assessing how pore-fluid pressure responds to applied stress over large scales in situ remains a challenging task; hence, spatial variations in response are not well understood, especially around active faults. Here I analyze previously unutilized records of dynamic strain and pore-pressure from regional and teleseismic earthquakes at Plate Boundary Observatory (PBO) stations from 2006 through 2012 to investigate variations in response along the Pacific/North American tectonic plate boundary. I find robust scaling-response coefficients between excess pore pressure and dynamic strain at each station that are spatially correlated: around the San Andreas and San Jacinto fault systems, the response is lowest in regions of the crust undergoing the highest rates of secular shear strain. PBO stations in the Parkfield instrument cluster are at comparable distances to the San Andreas fault (SAF), and spatial variations there follow patterns in dextral creep rates along the fault, with the highest response in the actively creeping section, which is consistent with a narrowing zone of strain accumulation seen in geodetic velocity profiles. At stations in the San Juan Bautista (SJB) and Anza instrument clusters, the response depends non-linearly on the inverse fault-perpendicular distance, with the response decreasing towards the fault; the SJB cluster is at the northern transition from creeping-to-locked behavior along the SAF, where creep rates are at moderate to low levels, and the Anza cluster is around the San Jacinto fault, where to date there have been no statistically significant creep rates observed at the surface. These results suggest that the strength

  13. Pore-forming activity and structural autoinhibition of the gasdermin family.

    PubMed

    Ding, Jingjin; Wang, Kun; Liu, Wang; She, Yang; Sun, Qi; Shi, Jianjin; Sun, Hanzi; Wang, Da-Cheng; Shao, Feng

    2016-07-01

    Inflammatory caspases cleave the gasdermin D (GSDMD) protein to trigger pyroptosis, a lytic form of cell death that is crucial for immune defences and diseases. GSDMD contains a functionally important gasdermin-N domain that is shared in the gasdermin family. The functional mechanism of action of gasdermin proteins is unknown. Here we show that the gasdermin-N domains of the gasdermin proteins GSDMD, GSDMA3 and GSDMA can bind membrane lipids, phosphoinositides and cardiolipin, and exhibit membrane-disrupting cytotoxicity in mammalian cells and artificially transformed bacteria. Gasdermin-N moved to the plasma membrane during pyroptosis. Purified gasdermin-N efficiently lysed phosphoinositide/cardiolipin-containing liposomes and formed pores on membranes made of artificial or natural phospholipid mixtures. Most gasdermin pores had an inner diameter of 10–14 nm and contained 16 symmetric protomers. The crystal structure of GSDMA3 showed an autoinhibited two-domain architecture that is conserved in the gasdermin family. Structure-guided mutagenesis demonstrated that the liposome-leakage and pore-forming activities of the gasdermin-N domain are required for pyroptosis. These findings reveal the mechanism for pyroptosis and provide insights into the roles of the gasdermin family in necrosis, immunity and diseases. PMID:27281216

  14. Effects of steam activation on the pore structure and surface chemistry of activated carbon derived from bamboo waste

    NASA Astrophysics Data System (ADS)

    Zhang, Yan-Juan; Xing, Zhen-Jiao; Duan, Zheng-Kang; Li, Meng; Wang, Yin

    2014-10-01

    The effects of steam activation on the pore structure evolution and surface chemistry of activated carbon (AC) obtained from bamboo waste were investigated. Nitrogen adsorption-desorption isotherms revealed that higher steam activation temperatures and/or times promoted the creation of new micropores and widened the existing micropores, consequently decreasing the surface area and total pore volume. Optimum conditions included an activation temperature of 850 °C, activation time of 120 min, and steam flush generated from deionized water of 0.2 cm3 min-1. Under these conditions, AC with a BET surface area of 1210 m2 g-1 and total pore volume of 0.542 cm-3 g-1was obtained. Changes in surface chemistry were determined through Boehm titration, pH measurement, Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). Results revealed the presence of a large number of basic groups on the surface of the pyrolyzed char and AC. Steam activation did not affect the species of oxygen-containing groups but changed the contents of these species when compared with pyrolyzed char. Scanning electron microscopy was used to observe the surface morphology of the products. AC obtained under optimum conditions showed a monolayer adsorption capacity of 330 mg g-1 for methylene blue (MB), which demonstrates its excellent potential for MB adsorption applications.

  15. Adsorption of carbon tetrachloride on graphitized thermal carbon black and in slit graphitic pores: five-site versus one-site potential models.

    PubMed

    Do, D D; Do, H D

    2006-05-18

    The performance of intermolecular potential models on the adsorption of carbon tetrachloride on graphitized thermal carbon black at various temperatures is investigated. This is made possible with the extensive experimental data of Machin and Ross(1), Avgul et al.,(2) and Pierce(3) that cover a wide range of temperatures. The description of all experimental data is only possible with the allowance for the surface mediation. If this were ignored, the grand canonical Monte Carlo (GCMC) simulation results would predict a two-dimensional (2D) transition even at high temperatures, while experimental data shows gradual change in adsorption density with pressure. In general, we find that the intermolecular interaction has to be reduced by 4% whenever particles are within the first layer close to the surface. We also find that this degree of surface mediation is independent of temperature. To understand the packing of carbon tetrachloride in slit pores, we compared the performance of the potential models that model carbon tetrachloride as either five interaction sites or one site. It was found that the five-site model performs better and describes the imperfect packing in small pores better. This is so because most of the strength of fluid-fluid interaction between two carbon tetrachloride molecules comes from the interactions among chlorine atoms. Methane, although having tetrahedral shape as carbon tetrachloride, can be effectively modeled as a pseudospherical particle because most of the interactions come from carbon-carbon interaction and hydrogen negligibly contributes to this. PMID:16686498

  16. Selective disruption of high sensitivity heat activation but not capsaicin activation of TRPV1 channels by pore turret mutations.

    PubMed

    Cui, Yuanyuan; Yang, Fan; Cao, Xu; Yarov-Yarovoy, Vladimir; Wang, KeWei; Zheng, Jie

    2012-04-01

    The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate.

  17. Pore size analysis of activated carbons from argon and nitrogen porosimetry using density functional theory

    SciTech Connect

    Dombrowski, R.J.; Hyduke, D.R.; Lastoskie, C.M.

    2000-05-30

    The authors present isotherms calculated from density functional theory for the adsorption of argon in model slit-shaped carbon pores at 77 K. The model isotherms are used to interpret experimental argon uptake measurements and to obtain the pore size distributions of several porous carbons. A similar set of density measurements and to obtain the pore size distributions of several porous carbons. A similar set of density functional theory isotherms, previously reported for nitrogen adsorption on carbon slit pores at 77 K, are used to determine pore size distributions for the same set of carbons. The pore size distribution maxima, mean pore widths, and specific pore volumes measured using the two different probe gases are all found to agree to within approximately 8% on average. Some of the differences in the pore size distributions obtained from argon and nitrogen porosimetry may be attributable to quadrupolar interactions of the nitrogen molecules with functional groups on the carbon surface.

  18. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores.

    PubMed

    Liu, Xing; Zhang, Zhibin; Ruan, Jianbin; Pan, Youdong; Magupalli, Venkat Giri; Wu, Hao; Lieberman, Judy

    2016-07-01

    Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1β. Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined. PMID:27383986

  19. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores.

    PubMed

    Liu, Xing; Zhang, Zhibin; Ruan, Jianbin; Pan, Youdong; Magupalli, Venkat Giri; Wu, Hao; Lieberman, Judy

    2016-07-06

    Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1β. Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined.

  20. Pore size distribution analysis of activated carbons prepared from coconut shell using methane adsorption data

    NASA Astrophysics Data System (ADS)

    Ahmadpour, A.; Okhovat, A.; Darabi Mahboub, M. J.

    2013-06-01

    The application of Stoeckli theory to determine pore size distribution (PSD) of activated carbons using high pressure methane adsorption data is explored. Coconut shell was used as a raw material for the preparation of 16 different activated carbon samples. Four samples with higher methane adsorption were selected and nitrogen adsorption on these adsorbents was also investigated. Some differences are found between the PSD obtained from the analysis of nitrogen adsorption isotherms and their PSD resulting from the same analysis using methane adsorption data. It is suggested that these differences may arise from the specific interactions between nitrogen molecules and activated carbon surfaces; therefore caution is required in the interpretation of PSD obtained from the nitrogen isotherm data.

  1. The Effect of Pore Connectivity on Water Adsorption Isotherms in Non-activated Graphitic Nanopores

    SciTech Connect

    StrioloDr., A; Gubbins, Dr. K. E.; Chialvo, Ariel A; Cummings, Peter T

    2005-01-01

    The adsorption of water in graphitic carbons is usually simulated via a weighted average of the adsorption isotherms simulated in carbon-slit pore of different widths. By following this procedure, details about pore morphology and pore connectivity may be overlooked. Towards a better match between virtual and real experiments, we present simulated adsorption isotherms for SPC/E model water in porous carbons composed by interconnected carbon-slit pores. The pores are separated from each other by one graphene layer. Imperfections (lack of carbon atoms) in the graphene layers result in interconnections between pores. The grand canonical Monte Carlo algorithm is used here to simulate water adsorption. Our results show that while the qualitative features obtained in the simulation of independent slit-shaped pores are reproduced when interconnected pores are considered, the adsorption isotherms rise more gradually and the adsorption/desorption hysteresis loops are narrower in the latter case.

  2. Sequence of Gating Charge Movement and Pore Gating in hERG Activation and Deactivation Pathways

    PubMed Central

    Goodchild, Samuel J.; Macdonald, Logan C.; Fedida, David

    2015-01-01

    KV11.1 voltage-gated K+ channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor movement, and also by a mode-shift after activation. To test these ideas, we compared the kinetics and voltage dependence of ionic activation and deactivation with gating charge movement. At 0 mV, gating charge moved ∼threefold faster than ionic current, which suggests the presence of additional slow transitions downstream of charge movement in the physiological activation pathway. A significant voltage sensor mode-shift was apparent by 24 ms at +60 mV in gating currents, and return of charge closely tracked pore closure after pulses of 100 and 300 ms duration. A deletion of the N-terminus PAS domain, mutation R4AR5A or the LQT2-causing mutation R56Q gave faster-deactivating channels that displayed an attenuated mode-shift of charge. This indicates that charge movement is perturbed by N- and C-terminus interactions, and that these domain interactions stabilize the open state and limit the rate of charge return. We conclude that slow on-gating charge movement can only partly account for slow hERG ionic activation, and that the rate of pore closure has a limiting role in the slow return of gating charges. PMID:25809256

  3. Changes in14c activity over time during vacuum distillation of carbon from rock pore water

    USGS Publications Warehouse

    Davidson, G.R.; Yang, I.C.

    1999-01-01

    The radiocarbon activity of carbon collected by vacuum distillation from a single partially saturated tuff began to decline after approximately 60% of the water and carbon had been extracted. Disproportionate changes in 14C activity and ??13C during distillation rule out simple isotopic fractionation as a causative explanation. Additional phenomena such as matrix diffusion and ion exclusion in micropores may play a role in altering the isotopic value of extracted carbon, but neither can fully account for the observed changes. The most plausible explanation is that distillation recovers carbon from an adsorbed phase that is depleted in 14C relative to DIC in the bulk pore water. ?? 1999 by the Arizona Board of Regents on behalf of the University of Arizona.

  4. Development and validation of pore structure models for adsorption in activated carbons

    SciTech Connect

    Davies, G.M.; Seaton, N.A.

    1999-09-14

    Predicting adsorption over a range of operating conditions and the improvement of the adsorbent itself are two important aspects that arise in the industrial application of adsorption. Both of these aspects can be addressed using molecular simulation techniques in conjunction with an appropriate model of the internal structure of the adsorbent. The internal structure of activated carbons is particularly difficult to model due to the fact that the structure is only locally crystalline and that most of the void volumes within the structure have length scales comparable to small molecules. This paper presents a systematic method to develop suitable models of the internal structure that are based on networks of regularly shaped model pores. Important aspects that are addressed include the realism and consistency of the resulting models. The method is illustrated using the adsorption of pure methane and ethane, and binary mixtures of these components, over a wide range of operating conditions onto four activated carbons.

  5. Intumescence and pore structure of alkali-activated volcanic glasses upon exposure to high temperatures

    NASA Astrophysics Data System (ADS)

    Erdogan, S. T.

    2015-12-01

    Structures formed with ground perlite, a natural volcanic glass, activated with NaOH solutions, are shown to possess the ability to expand up to ~225 % of their original volumes upon exposure to temperatures in the 200-600 °C range. Porous solid with 3-7 MPa compressive strength and ˜450 kg/m3 or higher density are obtained. The observed expansion is believed to occur due to a loss of silanol condensation water, as vapor and is accompanied by an up to ~20 % loss in mass. A drop in pH to near-neutral values supports this idea. The size and total amount of pores in the final solid are controlled by concentration of the NaOH solution and thermal processing conditions. The pores formed are observed to be ~1-10 μm to mm-sized. The ability of perlite-based solids to intumesce over specific temperature ranges could be beneficial in applications where absorption of thermal energy is necessary, such as passive fire protection.

  6. Geochemical controls on the composition of soil pore waters beneath a mixed waste disposal site in the unsaturated zone. [SNAKE RIVER PLAIN AQUIFER

    SciTech Connect

    Rawson, S.A.; Hubbell, J.M.

    1989-01-01

    Soil pore waters are collected routinely to monitor a thick unsaturated zone that separates a mixed waste disposal site containing transuranic and low-level radioactive wastes from the Snake River Plain aquifer. The chemistry of the soil pore waters has been studied to evaluate the possible control on the water composition by mineral equilibria and determine the extent, if any, of migration of radionuclides from the disposal site. Geochemical codes were used to perform speciation calculations for the waters. The results of speciation calculations suggest that the installation of the lysimeters affects the observed silica contents of the soil pore waters. The results also establish those chemical parameters that are controlled by secondary mineral precipitation. 15 refs., 6 figs., 1 tab.

  7. Pore-lining composition and capillary breakthrough pressure of mudstone caprocks : sealing efficiency at geologic CO2 storage sites.

    SciTech Connect

    Heath, Jason E.; Nemer, Martin B.; McPherson, Brian J. O. L.; Dewers, Thomas A.; Kotula, Paul Gabriel

    2010-12-01

    Subsurface containment of CO2 is predicated on effective caprock sealing. Many previous studies have relied on macroscopic measurements of capillary breakthrough pressure and other petrophysical properties without direct examination of solid phases that line pore networks and directly contact fluids. However, pore-lining phases strongly contribute to sealing behavior through interfacial interactions among CO2, brine, and the mineral or non-mineral phases. Our high resolution (i.e., sub-micron) examination of the composition of pore-lining phases of several continental and marine mudstones indicates that sealing efficiency (i.e., breakthrough pressure) is governed by pore shapes and pore-lining phases that are not identifiable except through direct characterization of pores. Bulk X-ray diffraction data does not indicate which phases line the pores and may be especially lacking for mudstones with organic material. Organics can line pores and may represent once-mobile phases that modify the wettability of an originally clay-lined pore network. For shallow formations (i.e., < {approx}800 m depth), interfacial tension and contact angles result in breakthrough pressures that may be as high as those needed to fracture the rock - thus, in the absence of fractures, capillary sealing efficiency is indicated. Deeper seals have poorer capillary sealing if mica-like wetting dominates the wettability. We thank the U.S. Department of Energy's National Energy Technology Laboratory and the Office of Basic Energy Sciences, and the Southeast and Southwest Carbon Sequestration Partnerships for supporting this work.

  8. Pore-lining composition and capillary breakthrough pressure of mudstone caprocks : sealing efficiency of geologic CO2 storage sites.

    SciTech Connect

    Petrusak, Robin; Heath, Jason E.; McPherson, Brian J. O. L.; Dewers, Thomas A.; Kotula, Paul Gabriel

    2010-08-01

    Subsurface containment of CO2 is predicated on effective caprock sealing. Many previous studies have relied on macroscopic measurements of capillary breakthrough pressure and other petrophysical properties without direct examination of solid phases that line pore networks and directly contact fluids. However, pore-lining phases strongly contribute to sealing behavior through interfacial interactions among CO2, brine, and the mineral or non-mineral phases. Our high resolution (i.e., sub-micron) examination of the composition of pore-lining phases of several continental and marine mudstones indicates that sealing efficiency (i.e., breakthrough pressure) is governed by pore shapes and pore-lining phases that are not identifiable except through direct characterization of pores. Bulk X-ray diffraction data does not indicate which phases line the pores and may be especially lacking for mudstones with organic material. Organics can line pores and may represent once-mobile phases that modify the wettability of an originally clay-lined pore network. For shallow formations (i.e., < {approx}800 m depth), interfacial tension and contact angles result in breakthrough pressures that may be as high as those needed to fracture the rock - thus, in the absence of fractures, capillary sealing efficiency is indicated. Deeper seals have poorer capillary sealing if mica-like wetting dominates the wettability.

  9. A macromolecular crowding study of RNA folding and activity: polymer pore size matters! (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Börner, Richard; Fiorini, Erica; Paudel, Bishnu; Rueda, David; Sigel, Roland K. O.

    2016-03-01

    Catalytic RNAs, like the group IIB intron ribozyme of S. cerevesiae, require a high magnesium(II) concentration to show folding and function in vitro [1]. In contrast, in vivo conditions are characterized by a highly crowded cellular environment and much lower ion concentration. Molecular crowding agents are a widespread tool to mimic cellular crowding [2]. However, particular physical/chemical properties explaining the crowders influence are mostly not understood. In this study, we gain new insights on how polymer properties like viscosity, pore size etc. influence the activity and folding of a large RNA. We combined bulk activity assays and single-molecule Förster Resonance Energy Transfer experiments, screening the PEG volume fraction (%) and molecular weight (MW). Our results revealed that upon the influence of crowding agents, a compaction of the underlying structure depends on the PEG % and the presence of different PEG MW and % unveiled an optimal pore size in terms of catalytic activity. In summary, an increasing density of the crowding environment shifts the RNA towards the most compact state, but the ribozyme is only active if the crowders network matches its size [4]. We interpret the most compact state as necessary, but not sufficient, to keep the ribozyme active. Financial support from the European Research Council (MIRNA N° 259092, to RKOS), the Swiss National Fund (SNF), and the Forschungskredit Grant of the University of Zürich (FK-14-096 and 15-092 to RB) are gratefully acknowledged. [1] Swisher J.F., Su L.J., Brenowitz M., Anderson V.E., Pyle A.M., J. Mol. Bio., 315, 297-310 (2002). [2] Kilburn D., Roh J.H., Guo L., Briber R.M., Woodson S.A., JACS, 132, 8690-6 (2010). [3] Steiner M., Karunatilaka K.S., Sigel R.K.O., Rueda D., Proc. Natl. Acad. Sci. U.S.A.,105, 13853-8 (2008). [4] aBörner R, Fiorini E, Sigel R.K.O., Chimia, 69, 207-212 (2015).; bFiorini E., Paudel B., Börner R., Rueda D., Sigel R.K.O., submitted. [5] König S.L.B., Hadzic M

  10. Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity

    PubMed Central

    Holden, Jennifer M.; Koreny, Ludek; Obado, Samson; Ratushny, Alexander V.; Chen, Wei-Ming; Chiang, Jung-Hsien; Kelly, Steven; Chait, Brian T.; Aitchison, John D.; Rout, Michael P.; Field, Mark C.

    2014-01-01

    The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle–dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina. PMID:24600046

  11. Branched pore kinetic model analysis of geosmin adsorption on super-powdered activated carbon.

    PubMed

    Matsui, Yoshihiko; Ando, Naoya; Sasaki, Hiroshi; Matsushita, Taku; Ohno, Koichi

    2009-07-01

    Super-powdered activated carbon (S-PAC) is activated carbon of much finer particle size than powdered activated carbon (PAC). Geosmin is a naturally occurring taste and odor compound that impairs aesthetic quality in drinking water. Experiments on geosmin adsorption on S-PAC and PAC were conducted, and the results using adsorption kinetic models were analyzed. PAC pulverization, which produced the S-PAC, did not change geosmin adsorption capacity, and geosmin adsorption capacities did not differ between S-PAC and PAC. Geosmin adsorption kinetics, however, were much higher on S-PAC than on PAC. A solution to the branched pore kinetic model (BPKM) was developed, and experimental adsorption kinetic data were analyzed by BPKM and by a homogeneous surface diffusion model (HSDM). The HSDM describing the adsorption behavior of geosmin required different surface diffusivity values for S-PAC and PAC, which indicated a decrease in surface diffusivity apparently associated with activated carbon particle size. The BPKM, consisting of macropore diffusion followed by mass transfer from macropore to micropore, successfully described the batch adsorption kinetics on S-PAC and PAC with the same set of model parameter values, including surface diffusivity. The BPKM simulation clearly showed geosmin removal was improved as activated carbon particle size decreased. The simulation also implied that the rate-determining step in overall mass transfer shifted from intraparticle radial diffusion in macropores to local mass transfer from macropore to micropore. Sensitivity analysis showed that adsorptive removal of geosmin improved with decrease in activated carbon particle size down to 1microm, but further particle size reduction produced little improvement.

  12. New general pore size distribution model by classical thermodynamics application: Activated carbon

    USGS Publications Warehouse

    Lordgooei, M.; Rood, M.J.; Rostam-Abadi, M.

    2001-01-01

    A model is developed using classical thermodynamics to characterize pore size distributions (PSDs) of materials containing micropores and mesopores. The thermal equation of equilibrium adsorption (TEEA) is used to provide thermodynamic properties and relate the relative pore filling pressure of vapors to the characteristic pore energies of the adsorbent/adsorbate system for micropore sizes. Pore characteristic energies are calculated by averaging of interaction energies between adsorbate molecules and adsorbent pore walls as well as considering adsorbate-adsorbate interactions. A modified Kelvin equation is used to characterize mesopore sizes by considering variation of the adsorbate surface tension and by excluding the adsorbed film layer for the pore size. The modified-Kelvin equation provides similar pore filling pressures as predicted by density functional theory. Combination of these models provides a complete PSD of the adsorbent for the micropores and mesopores. The resulting PSD is compared with the PSDs from Jaroniec and Choma and Horvath and Kawazoe models as well as a first-order approximation model using Polanyi theory. The major importance of this model is its basis on classical thermodynamic properties, less simplifying assumptions in its derivation compared to other methods, and ease of use.

  13. Sulfur infiltrated activated carbon cathodes for lithium sulfur cells: The combined effects of pore size distribution and electrolyte molarity

    NASA Astrophysics Data System (ADS)

    Lee, Jung Tae; Zhao, Youyang; Kim, Hyea; Cho, Won Il; Yushin, Gleb

    2014-02-01

    In this paper, we adopted three different commercial activated carbon samples (ACs) having different particle size, specific surface area and pore size to make sulfur-carbon (S-AC) nanocomposites for rechargeable lithium sulfur batteries. The effect of the physical parameters of ACs and the combined effect of electrolyte molarity were investigated. The performance of the cells at two different temperatures of 25 and 70 °C were compared. For room temperature operation of the cells, the capacities of S infiltrated into microporous AC having smaller pore size and stronger interactions with sulfur and sulfides were lower than those of the S infiltrated into micro- and mesoporous ACs containing larger pores. In contrast, the microporous AC demonstrated higher capacity at the elevated temperature due to the improved ion transport rate. The effect of electrolyte molarity on the performance of Li/S cells was found to depend on the AC pore size and particle size distributions. Increasing electrolyte molarity from 1 to 3 M demonstrated improved cell performance and reduced polysulfide dissolution in all the studied S-AC samples. However, further increasing electrolyte salt concentration resulted in a high polarization and reduced cell performance in S-ACs having large particle size or smaller pores.

  14. Nuclear pore protein NUP88 activates anaphase-promoting complex to promote aneuploidy.

    PubMed

    Naylor, Ryan M; Jeganathan, Karthik B; Cao, Xiuqi; van Deursen, Jan M

    2016-02-01

    The nuclear pore complex protein NUP88 is frequently elevated in aggressive human cancers and correlates with reduced patient survival; however, it is unclear whether and how NUP88 overexpression drives tumorigenesis. Here, we show that mice overexpressing NUP88 are cancer prone and form intestinal tumors. To determine whether overexpression of NUP88 drives tumorigenesis, we engineered transgenic mice with doxycycline-inducible expression of Nup88. Surprisingly, NUP88 overexpression did not alter global nuclear transport, but was a potent inducer of aneuploidy and chromosomal instability. We determined that NUP88 and the nuclear transport factors NUP98 and RAE1 comprise a regulatory network that inhibits premitotic activity of the anaphase-promoting complex/cyclosome (APC/C). When overexpressed, NUP88 sequesters NUP98-RAE1 away from APC/CCDH1, triggering proteolysis of polo-like kinase 1 (PLK1), a tumor suppressor and multitasking mitotic kinase. Premitotic destruction of PLK1 disrupts centrosome separation, causing mitotic spindle asymmetry, merotelic microtubule-kinetochore attachments, lagging chromosomes, and aneuploidy. These effects were replicated by PLK1 insufficiency, indicating that PLK1 is responsible for the mitotic defects associated with NUP88 overexpression. These findings demonstrate that the NUP88-NUP98-RAE1-APC/CCDH1 axis contributes to aneuploidy and suggest that it may be deregulated in the initiating stages of a broad spectrum of human cancers. PMID:26731471

  15. Nuclear pore protein NUP88 activates anaphase-promoting complex to promote aneuploidy

    PubMed Central

    Naylor, Ryan M.; Jeganathan, Karthik B.; Cao, Xiuqi; van Deursen, Jan M.

    2016-01-01

    The nuclear pore complex protein NUP88 is frequently elevated in aggressive human cancers and correlates with reduced patient survival; however, it is unclear whether and how NUP88 overexpression drives tumorigenesis. Here, we show that mice overexpressing NUP88 are cancer prone and form intestinal tumors. To determine whether overexpression of NUP88 drives tumorigenesis, we engineered transgenic mice with doxycycline-inducible expression of Nup88. Surprisingly, NUP88 overexpression did not alter global nuclear transport, but was a potent inducer of aneuploidy and chromosomal instability. We determined that NUP88 and the nuclear transport factors NUP98 and RAE1 comprise a regulatory network that inhibits premitotic activity of the anaphase-promoting complex/cyclosome (APC/C). When overexpressed, NUP88 sequesters NUP98-RAE1 away from APC/CCDH1, triggering proteolysis of polo-like kinase 1 (PLK1), a tumor suppressor and multitasking mitotic kinase. Premitotic destruction of PLK1 disrupts centrosome separation, causing mitotic spindle asymmetry, merotelic microtubule-kinetochore attachments, lagging chromosomes, and aneuploidy. These effects were replicated by PLK1 insufficiency, indicating that PLK1 is responsible for the mitotic defects associated with NUP88 overexpression. These findings demonstrate that the NUP88-NUP98-RAE1-APC/CCDH1 axis contributes to aneuploidy and suggest that it may be deregulated in the initiating stages of a broad spectrum of human cancers. PMID:26731471

  16. Neutralization of a single arginine residue gates open a two-pore domain, alkali-activated K+ channel

    PubMed Central

    Niemeyer, María Isabel; González-Nilo, Fernando D.; Zúñiga, Leandro; González, Wendy; Cid, L. Pablo; Sepúlveda, Francisco V.

    2007-01-01

    Potassium channels share a common selectivity filter that determines the conduction characteristics of the pore. Diversity in K+ channels is given by how they are gated open. TASK-2, TALK-1, and TALK-2 are two-pore region (2P) KCNK K+ channels gated open by extracellular alkalinization. We have explored the mechanism for this alkalinization-dependent gating using molecular simulation and site-directed mutagenesis followed by functional assay. We show that the side chain of a single arginine residue (R224) near the pore senses pH in TASK-2 with an unusual pKa of 8.0, a shift likely due to its hydrophobic environment. R224 would block the channel through an electrostatic effect on the pore, a situation relieved by its deprotonation by alkalinization. A lysine residue in TALK-2 fulfills the same role but with a largely unchanged pKa, which correlates with an environment that stabilizes its positive charge. In addition to suggesting unified alkaline pH-gating mechanisms within the TALK subfamily of channels, our results illustrate in a physiological context the principle that hydrophobic environment can drastically modulate the pKa of charged amino acids within a protein. PMID:17197424

  17. Pore-water extraction from unsaturated tuff by triaxial and one-dimensional compression methods, Nevada Test Site, Nevada

    USGS Publications Warehouse

    Mower, Timothy E.; Higgins, Jerry D.; Yang, In C.; Peters, Charles A.

    1994-01-01

    Study of the hydrologic system at Yucca Mountain, Nevada, requires the extraction of pore-water samples from welded and nonwelded, unsaturated tuffs. Two compression methods (triaxial compression and one-dimensional compression) were examined to develop a repeatable extraction technique and to investigate the effects of the extraction method on the original pore-fluid composition. A commercially available triaxial cell was modified to collect pore water expelled from tuff cores. The triaxial cell applied a maximum axial stress of 193 MPa and a maximum confining stress of 68 MPa. Results obtained from triaxial compression testing indicated that pore-water samples could be obtained from nonwelded tuff cores that had initial moisture contents as small as 13 percent (by weight of dry soil). Injection of nitrogen gas while the test core was held at the maximum axial stress caused expulsion of additional pore water and reduced the required initial moisture content from 13 to 11 percent. Experimental calculations, together with experience gained from testing moderately welded tuff cores, indicated that the triaxial cell used in this study could not apply adequate axial or confining stress to expel pore water from cores of densely welded tuffs. This concern led to the design, fabrication, and testing of a one-dimensional compression cell. The one-dimensional compression cell used in this study was constructed from hardened 4340-alloy and nickel-alloy steels and could apply a maximum axial stress of 552 MPa. The major components of the device include a corpus ring and sample sleeve to confine the sample, a piston and base platen to apply axial load, and drainage plates to transmit expelled water from the test core out of the cell. One-dimensional compression extracted pore water from nonwelded tuff cores that had initial moisture contents as small as 7.6 percent; pore water was expelled from densely welded tuff cores that had initial moisture contents as small as 7

  18. Use of activated carbon as a support medium for H2S biofiltration and effect of bacterial immobilization on available pore surface.

    PubMed

    Ng, Y L; Yan, R; Chen, X G; Geng, A L; Gould, W D; Liang, D T; Koe, L C C

    2004-12-01

    The use of support media for the immobilization of microorganisms is widely known to provide a surface for microbial growth and a shelter that protects the microorganisms from inhibitory compounds. In this study, activated carbon is used as a support medium for the immobilization of microorganisms enriched from municipal sewage activated sludge to remove gas-phase hydrogen sulfide (H2S), a major odorous component of waste gas from sewage treatment plants. A series of designed experiments is used to examine the effect on bacteria-immobilized activated carbon (termed "biocarbon") due to physical adsorption, chemical reaction, and microbial degradation in the overall removal of H2S. H2S breakthrough tests are conducted with various samples, including microbe-immobilized carbon and Teflon discs, salts-medium-washed carbon, and ultra-pure water-washed carbon. The results show a higher removal capacity for the microbe-immobilized activated carbon compared with the activated carbon control in a batch biofilter column. The increase in removal capacity is attributed to the role played by the immobilized microorganisms in metabolizing adsorbed sulfur and sulfur compounds on the biocarbon, hence releasing the adsorption sites for further H2S uptake. The advantage for activated carbon serving as the support medium is to adsorb a high initial concentration of substrate and progressively release this for microbial degradation, hence acting as a buffer for the microorganisms. Results obtained from surface area and pore size distribution analyses of the biocarbon show a correlation between the available surface area and pore volume with the extent of microbial immobilization and H2S uptake. The depletion of surface area and pore volume is seen as one of the factors which cause the onset of column breakthrough. Microbial growth retardation is due to the accumulation of metabolic products (i.e., sulfuric acid); and a lack of water and nutrient salts in the batch biofilter are other

  19. Isolating the effect of pore size distribution on electrochemical double-layer capacitance using activated fluid coke

    NASA Astrophysics Data System (ADS)

    Zuliani, Jocelyn E.; Tong, Shitang; Kirk, Donald W.; Jia, Charles Q.

    2015-12-01

    Electrochemical double-layer capacitors (EDLCs) use physical ion adsorption in the capacitive electrical double layer of high specific surface area (SSA) materials to store electrical energy. Previous work shows that the SSA-normalized capacitance increases when pore diameters are less than 1 nm. However, there still remains uncertainty about the charge storage mechanism since the enhanced SSA-normalized capacitance is not observed in all microporous materials. In previous studies, the total specific surface area and the chemical composition of the electrode materials were not controlled. The current work is the first reported study that systematically compares the performance of activated carbon prepared from the same raw material, with similar chemical composition and specific surface area, but different pore size distributions. Preparing samples with similar SSAs, but different pores sizes is not straightforward since increasing pore diameters results in decreasing the SSA. This study observes that the microporous activated carbon has a higher SSA-normalized capacitance, 14.1 μF cm-2, compared to the mesoporous material, 12.4 μF cm-2. However, this enhanced SSA-normalized capacitance is only observed above a threshold operating voltage. Therefore, it can be concluded that a minimum applied voltage is required to induce ion adsorption in these sub-nanometer micropores, which increases the capacitance.

  20. Pore structure effects on Ca-based sorbent sulfation capacity at medium temperatures: activated carbon as sorbent/catalyst support.

    PubMed

    Tseng, Hui-Hsin; Wey, Ming-Yen; Lin, Chiou-Liang; Chang, Yu-Chen

    2002-11-01

    The reaction between three different Ca-based sorbents and SO2 were studied in a medium temperature range (473-773 K). The largest SO2 capture was found with Ca(OH)2 at 773 K, 126.31 mg SO2 x g Ca(OH)2(-1), and the influence of SO2 concentration on the sorbent utilization was observed. Investigations of the internal porous structure of Ca-based sorbents showed that the initial reaction rate was controlled by the surface area, and once the sulfated products were produced, pore structure dominated. To increase the surface area of Ca-based sorbents available to interact with and retain SO2, one kind of CaO/ activated carbon (AC) sorbent/catalyst was prepared to study the effect of AC on the dispersion of Ca-based materials. The results indicated that the Ca-based material dispersed on high-surface-area AC had more capacities for SO2 than unsupported Ca-based sorbents. The initial reaction rates of the reaction between SO2 and Ca-based sorbents and the prepared CaO/AC sorbents/catalysts were measured. Results showed that the reaction rate apparently increased with the presence of AC. It was concluded that CaO/AC was the active material in the desulfurization reaction. AC acting as the support can play a role to supply O2 to increase the affinity to SO2. Moreover, when AC is acting as a support, the surface oxygen functional group formed on the surface of AC can serve as a new site for SO2 adsorption.

  1. Pneumolysin Activates Macrophage Lysosomal Membrane Permeabilization and Executes Apoptosis by Distinct Mechanisms without Membrane Pore Formation

    PubMed Central

    Bewley, Martin A.; Naughton, Michael; Preston, Julie; Mitchell, Andrea; Holmes, Ashleigh; Marriott, Helen M.; Read, Robert C.; Mitchell, Timothy J.; Whyte, Moira K. B.

    2014-01-01

    ABSTRACT Intracellular killing of Streptococcus pneumoniae is complemented by induction of macrophage apoptosis. Here, we show that the toxin pneumolysin (PLY) contributes both to lysosomal/phagolysosomal membrane permeabilization (LMP), an upstream event programing susceptibility to apoptosis, and to apoptosis execution via a mitochondrial pathway, through distinct mechanisms. PLY is necessary but not sufficient for the maximal induction of LMP and apoptosis. PLY’s ability to induce both LMP and apoptosis is independent of its ability to form cytolytic pores and requires only the first three domains of PLY. LMP involves TLR (Toll-like receptor) but not NLRP3/ASC (nucleotide-binding oligomerization domain [Nod]-like receptor family, pyrin domain-containing protein 3/apoptosis-associated speck-like protein containing a caspase recruitment domain) signaling and is part of a PLY-dependent but phagocytosis-independent host response that includes the production of cytokines, including interleukin-1 beta (IL-1β). LMP involves progressive and selective permeability to 40-kDa but not to 250-kDa fluorescein isothiocyanate (FITC)-labeled dextran, as PLY accumulates in the cytoplasm. In contrast, the PLY-dependent execution of apoptosis requires phagocytosis and is part of a host response to intracellular bacteria that also includes NO generation. In cells challenged with PLY-deficient bacteria, reconstitution of LMP using the lysomotrophic detergent LeuLeuOMe favored cell necrosis whereas PLY reconstituted apoptosis. The results suggest that PLY contributes to macrophage activation and cytokine production but also engages LMP. Following bacterial phagocytosis, PLY triggers apoptosis and prevents macrophage necrosis as a component of a broad-based antimicrobial strategy. This illustrates how a key virulence factor can become the focus of a multilayered and coordinated innate response by macrophages, optimizing pathogen clearance and limiting inflammation. PMID:25293758

  2. Effect of pore blockage on adsorption isotherms and dynamics: Anomalous adsorption of iodine on activated carbon

    SciTech Connect

    Bhatia, S.K.; Liu, F.; Arvind, G.

    2000-04-18

    Isotherm hysteresis and pore-clocking effects of trapped molecules on adsorption dynamics is studied here, using the iodine-carbon system in the 300--343 K temperature range. It is found that a portion of the iodine is strongly adsorbed, and does not desorb, even over very long time scales, while the remainder adsorbs reversibly as a homogeneous monolayer with a Langmuirian isotherm in mesopores. The strongly adsorbed iodine appears to adsorb in micropores and at the mesopore mouths, hindering uptake of the reversible iodine. The uptake data for the adsorption and desorption dynamics of the reversible part is found to be best explained by means of a pore mouth resistance control mechanism. it is concluded that the dynamics of the adsorption and desorption at the pore mouth is important at early stages of the process.

  3. The Mg isotopic composition of marine pore fluids from ODP Site 807A (Ontong Java Plateau): Implications for the Cenozoic Mg chemistry of the ocean

    NASA Astrophysics Data System (ADS)

    Fantle, M. S.; Teng, F.

    2011-12-01

    The Mg isotopic compositions (δ26Mg, relative to DSM3) of pore fluids from ODP Leg 130, Site 807A, as measured by MC-ICP-MS, are found to be between -0.25 and -0.79% (ave: -0.55%). The shallowest pore fluid measured was sampled at 13.4 mbsf while the deepest was sampled at 738 mbsf (a time span of ~35 Ma); the total section thickness is ~1350 meters. The sedimentary section is carbonate-rich, with a hole average CaCO3 content of ~92 wt%, and overlies basaltic basement. There is a systematic decrease in δ26Mg from the top of the section (~ -0.8%) to deeper in the section (~ -0.3%) that coincides with a decrease in pore fluid Mg and an increase in pore fluid Ca concentrations. The δ26Mg values at the upper and lower boundaries of the column are consistent with the isotopic composition of laboratory seawater (-0.84 ± 0.10%) and Kilbourne Hole olivine (-0.26 ± 0.05%) standards, respectively, the latter of which is isotopically identical to fresh basalt. The most basic interpretation of the data is that the pore fluids at 807A reflect modern seawater δ26Mg at the top of the section and basement basaltic Mg at ~800 mbsf. Because the section contains ~550 meters of carbonate below the deepest pore fluid measured, this implies a strong basement influence on Mg isotopic composition well above the sediment-basement interface. That the bulk carbonate and pore fluid are out of equilibrium is an expected result, based on the large reactive length scale at 807A (~1500 m) [1-2]. Consequently, because the previously measured δ26Mg values of bulk carbonates at 807A are ~ -4 to -5%, there is long-term leverage to change the isotopic composition of carbonates over time scales of tens of millions of years if we assume that the fractionation factor during diagenesis is different from ~0.9963 (the value assumed for biogenic carbonate formed in the surface ocean). Unlike Ca isotopes, the pore fluid is not well buffered with respect to Mg over geological time scales. Therefore, if

  4. Cytolytic and antibacterial activity of synthetic peptides derived from amoebapore, the pore-forming peptide of Entamoeba histolytica.

    PubMed Central

    Leippe, M; Andrä, J; Müller-Eberhard, H J

    1994-01-01

    The pore-forming peptide amoebapore is considered part of the cytolytic armament of pathogenic Entamoeba histolytica. Amoebapore is composed of 77 amino acid residues arranged in four alpha-helical domains. For structure-function analysis, synthetic peptides were constructed corresponding to these four domains: H1 (residues 1-22), H2 (25-39), H3 (40-64), and H4 (67-77). The peptides H1 and H3, representing two highly amphipathic alpha-helical regions of amoebapore, possessed pore-forming activity. Peptide H3 displayed cytolytic and antibacterial functions similar to those of natural amoebapore. The most potent antibacterial activity and the broadest activity spectrum were expressed by H1-Mel, a hybrid molecule composed of the N-terminal alpha-helix of amoebapore and the C-terminal hexapeptide of melittin from the venom of Apis mellifera. Images PMID:8146160

  5. Preparation of Granular Red Mud Adsorbent using Different Binders by Microwave Pore - Making and Activation Method

    NASA Astrophysics Data System (ADS)

    Le, Thiquynhxuan; Wang, Hanrui; Ju, Shaohua; Peng, Jinhui; Zhou, Liexing; Wang, Shixing; Yin, Shaohua; Liu, Chao

    2016-04-01

    In this work, microwave energy is used for preparing a granular red mud (GRM) adsorbent made of red mud with different binders, such as starch, sodium silicate and cement. The effects of the preparation parameters, such as binder type, binder addition ratio, microwave heating temperature, microwave power and holding time, on the absorption property of GRM are investigated. The BET surface area, strength, pore structure, XRD and SEM of the GRM absorbent are analyzed. The results show that the microwave roasting has a good effect on pore-making of GRM, especially when using organic binder. Both the BET surface area and the strength of GRM obtained by microwave heating are significantly higher than that by conventional heating. The optimum conditions are obtained as follows: 6:100 (w/w) of starch to red mud ratio, microwave roasting with a power of 2.6 kW at 500℃ for holding time of 30 min. The BET surface area, pore volume and average pore diameter of GRM prepared at the optimum conditions are 15.58 m2/g, 0.0337 cm3/g and 3.1693 A0, respectively.

  6. Catalysis: Elusive active site in focus

    NASA Astrophysics Data System (ADS)

    Labinger, Jay A.

    2016-08-01

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

  7. Composition of pore water in lake sediments, research site "B", Osage County, Oklahoma: Implications for lake water quality and benthic organisms

    USGS Publications Warehouse

    Zielinski, R.A.; Herkelrath, W.N.; Otton, J.K.

    2007-01-01

    Shallow ground water at US Geological Survey research site B in northeastern Oklahoma is contaminated with NaCl-rich brine from past and present oil production operations. Contaminated ground water provides a potential source of salts, metals, and hydrocarbons to sediment and water of adjacent Skiatook Lake. A former brine storage pit 10 m in diameter that is now submerged just offshore from site B provides an additional source of contamination. Cores of the upper 16-40 cm of lake sediment were taken at the submerged brine pit, near an offshore saline seep, and at a location containing relatively uncontaminated lake sediment. Pore waters from each 2-cm interval were separated by centrifugation and analyzed for dissolved anions, cations, and trace elements. High concentrations of dissolved Cl- in pore waters (200-5000 mg/L) provide the most direct evidence of contamination, and contrast sharply with an average value of only about 37 mg/L in Skiatook Lake. Chloride/Br- mass ratios of 220-240 in contaminated pore waters are comparable to values in contaminated well waters collected onshore. Dissolved concentrations of Se, Pb, Cu and Ni in Cl--rich pore waters exceed current US Environmental Protection Agency criteria for probable toxicity to aquatic life. At the submerged brine storage pit, the increase of Cl- concentration with depth is consistent with diffusion-dominant transport from deeper contaminated sediments. Near the offshore saline seep, pore water Cl- concentrations are consistently high and vary irregularly with depth, indicating probable Cl- transport by layer-directed advective flow. Estimated annual contributions of Cl- to the lake from the brine storage pit (???20 kg) and the offshore seep (???9 kg) can be applied to any number of similar sources. Generous estimates of the number of such sources at site B indicate minimal impact on water quality in the local inlet of Skiatook Lake. Similar methodologies can be applied at other sites of Na

  8. Effects of pore structure and electrolyte on the capacitive characteristics of steam- and KOH-activated carbons for supercapacitors

    NASA Astrophysics Data System (ADS)

    Wu, Feng-Chin; Tseng, Ru-Ling; Hu, Chi-Chang; Wang, Chen-Ching

    Four kinds of activated carbons (denoted as ACs) with specific surface area of ca. 1050 m 2 g -1 were fabricated from fir wood and pistachio shell by means of steam activation or chemical activation with KOH. Pore structures of ACs were characterized by a t-plot method based on N 2 adsorption isotherms. The amount of mesopores within KOH-activated carbons ranged from 9.2 to 15.3% while 33.3-49.5% of mesopores were obtained for the steam-activated carbons. The pore structure, surface functional groups, and raw materials of ACs, as well as pH and the supporting electrolyte were also found to be significant factors determining the capacitive characteristics of ACs. The excellent capacitive characteristics in both acidic and neutral media and the weak dependence of the specific capacitance on the scan rate of cyclic voltammetry (CV) for the ACs derived from the pistachio shell with steam activation (denoted as P-H 2O-AC) revealed their promising potential in the application of supercapacitors. The ACs derived from fir wood with KOH activation (denoted as F-KOH-AC), on the other hand, showed the best capacitive performance in H 2SO 4 due to excellent reversibility and high specific capacitance (180 F g -1 measured at 10 mV s -1), which is obviously larger than 100 F g -1 (a typical value of activated carbons with specific surface areas equal to/above 1000 m 2 g -1).

  9. Predicting pore pressure in active fold-thrust systems: An empirical model for the deepwater Sabah foldbelt

    NASA Astrophysics Data System (ADS)

    Couzens-Schultz, Brent A.; Azbel, Konstantin

    2014-12-01

    Measurements related to mudrock (shale and siltstone) porosity such as acoustic velocity, density or electrical resistivity, have traditionally been used to predict pore pressures in extensional stress settings. The underlying assumption is that burial and vertical effective stress (VES), which is the overburden minus the pore pressure, controls the compaction of these rocks through porosity loss. The dataset presented here compares VES and acoustic velocity of similar composition mudrocks in both an extensional and a compressional stress setting. In the extensional stress environment, the mudrocks follow a typical compaction trend with a porosity loss and increase in acoustic velocity that can be related to VES. In an active fold-thrust belt, the compressive stresses further reduce the porosity and increase the acoustic velocity of the mudrocks. First a layer-parallel shortening compacts sediments beyond what is observed for the VES. This additional compaction is further enhanced near thrust faults and in anticlinal forelimbs, presumably due to additional shear stress in these areas. The mudrocks located in folds that are buried by additional sedimentation do not compact again until the tectonic compaction is overridden by enough new burial. After that, the mudrocks follow the observed extensional setting compaction trend. In the fold-thrust belt, the observed reduction in porosity by stresses other than burial leads to an under-prediction of pore pressure using traditional methods. To account for this, we present a correction that can be applied to the acoustic velocity (or porosity) using two parameters: (a) proximity to thrust faults and anticlinal forelimbs and (b) the amount of burial after fold formation. With these corrections, the extensional velocity-VES compaction trend can be used to accurately predict pore pressure within the active fold-thrust belt. The correction is calibrated with well data and is empirical. None-the-less, it is a first step toward

  10. Estimation of activation energy for electroporation and pore growth rate in liquid crystalline and gel phases of lipid bilayers using molecular dynamics simulations.

    PubMed

    Majhi, Amit Kumar; Kanchi, Subbarao; Venkataraman, V; Ayappa, K G; Maiti, Prabal K

    2015-11-28

    Molecular dynamics simulations of electroporation in POPC and DPPC lipid bilayers have been carried out at different temperatures ranging from 230 K to 350 K for varying electric fields. The dynamics of pore formation, including threshold field, pore initiation time, pore growth rate, and pore closure rate after the field is switched off, was studied in both the gel and liquid crystalline (Lα) phases of the bilayers. Using an Arrhenius model of pore initiation kinetics, the activation energy for pore opening was estimated to be 25.6 kJ mol(-1) and 32.6 kJ mol(-1) in the Lα phase of POPC and DPPC lipids respectively at a field strength of 0.32 V nm(-1). The activation energy decreases to 24.2 kJ mol(-1) and 23.7 kJ mol(-1) respectively at a higher field strength of 1.1 V nm(-1). At temperatures below the melting point, the activation energy in the gel phase of POPC and DPPC increases to 28.8 kJ mol(-1) and 34.4 kJ mol(-1) respectively at the same field of 1.1 V nm(-1). The pore closing time was found to be higher in the gel than in the Lα phase. The pore growth rate increases linearly with temperature and quadratically with field, consistent with viscosity limited growth models.

  11. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

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

  12. Synthesis of pore-variable mesoporous CdS and evaluation of its photocatalytic activity in degrading methylene blue

    SciTech Connect

    Zhang, Wei-Min; Jiang, Yao-Quan; Cao, Xiao-Yan; Chen, Meng; Ge, Dong-Lai; Sun, Zhong-Xi

    2013-10-15

    Graphical abstract: - Highlights: • Self-templated synthesis of tubular CdS. • Cadmium complexes of aliphatic acids sustain the network of mesoporous structures. • Aliphatic acids affect the phase composition and particle size. • Pore size and volume vary with aliphatic acids having different hydrocarbonyl. - Abstract: In this study, mesoporous CdS polycrystallites have been synthesized using aliphatic acids of hexanoic acid, octanoic acid, and oleic acid as coordinating and capping agents, respectively. The fibrous Cd–fatty acid salts act as a template to form the tubular CdS. The organic species are found to be necessary for maintaining the network of mesoporous CdS. The characterization results indicate that the shorter carbon chain length in aliphatic acids favors the wurtzite phase and particle size growth the specific surface area, pore diameter and pore volume show a monotonic raise with increasing carbon chain. The photocatalytic activities of mesoporous CdS tubes exhibit much higher efficiency than those of nanosized CdS powders in decolorizing methylene blue under simulated visible light.

  13. An amino acid outside the pore region influences apamin sensitivity in small conductance Ca2+-activated K+ channels.

    PubMed

    Nolting, Andreas; Ferraro, Teresa; D'hoedt, Dieter; Stocker, Martin

    2007-02-01

    Small conductance calcium-activated potassium channels (SK, K(Ca)) are a family of voltage-independent K+ channels with a distinct physiology and pharmacology. The bee venom toxin apamin inhibits exclusively the three cloned SK channel subtypes (SK1, SK2, and SK3) with different affinity, highest for SK2, lowest for SK1, and intermediate for SK3 channels. The high selectivity of apamin made it a valuable tool to study the molecular makeup and function of native SK channels. Three amino acids located in the outer vestibule of the pore are of particular importance for the different apamin sensitivities of SK channels. Chimeric SK1 channels, enabling the homomeric expression of the rat SK1 (rSK1) subunit and containing the core domain (S1-S6) of rSK1, are apamin-insensitive. By contrast, channels formed by the human orthologue human SK1 (hSK1) are sensitive to apamin. This finding hinted at the involvement of regions beyond the pore as determinants of apamin sensitivity, because hSK1 and rSK1 have an identical amino acid sequence in the pore region. Here we investigated which parts of the channels outside the pore region are important for apamin sensitivity by constructing chimeras between apamin-insensitive and -sensitive SK channel subunits and by introducing point mutations. We demonstrate that a single amino acid situated in the extracellular loop between the transmembrane segments S3 and S4 has a major impact on apamin sensitivity. Our findings enabled us to convert the hSK1 channel into a channel that was as sensitive for apamin as SK2, the SK channel with the highest sensitivity.

  14. Fungicidal effect of piscidin on Candida albicans: pore formation in lipid vesicles and activity in fungal membranes.

    PubMed

    Sung, Woo Sang; Lee, Juneyoung; Lee, Dong Gun

    2008-10-01

    Piscidin, a 22-residue cationic peptide isolated from the mast cells of hybrid striped bass, has potent antimicrobial activities. In the present study, we investigated the fungicidal activity and mode of action of piscidins. Fungicidal and hemolytic assays were examined in order to assess their potency and toxicity, respectively. The results showed that fungicidal and hemolytic activities were higher for piscidin 1 (P1) than piscidin 3 (P3). Additionally, the abilities to permeabilize the model phospholipids membranes were also higher for P1 than P3, which were consistent with the biological activities of P1 and P3. These results suggest that the biological action of the peptides may be carried out through the lipid membrane. To understand the fungicidal properties of P1, we focused on a membrane-active mechanism of the peptide by in vivo testing against Candida albicans as the model organism. Flow cytometric analysis by using bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4)(3)] and protoplast regeneration experiments showed that P1 caused fungal membrane damage. Furthermore, fluorescence analysis, using 1,6-diphenyl-1,3,5-hexatriene, revealed that these peptides created pores in fungal membranes. Thus, the present study demonstrated that piscidins exert their fungicidal effects by disrupting fungal membrane through pore formation.

  15. Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD.

    PubMed

    Chatterjee, Abhishek; Caballero-Franco, Celia; Bakker, Dannika; Totten, Stephanie; Jardim, Armando

    2015-10-16

    Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280-320-kDa oligomeric structure consisting of ∼6-7 subunits.

  16. Immobilizing highly catalytically active Pt nanoparticles inside the pores of metal-organic framework: a double solvents approach.

    PubMed

    Aijaz, Arshad; Karkamkar, Abhi; Choi, Young Joon; Tsumori, Nobuko; Rönnebro, Ewa; Autrey, Tom; Shioyama, Hiroshi; Xu, Qiang

    2012-08-29

    Ultrafine Pt nanoparticles were successfully immobilized inside the pores of a metal-organic framework, MIL-101, without aggregation of Pt nanoparticles on the external surfaces of framework by using a "double solvents" method. TEM and electron tomographic measurements clearly demonstrated the uniform three-dimensional distribution of the ultrafine Pt NPs throughout the interior cavities of MIL-101. The resulting Pt@MIL-101 composites represent the first highly active MOF-immobilized metal nanocatalysts for catalytic reactions in all three phases: liquid-phase ammonia borane hydrolysis, solid-phase ammonia borane thermal dehydrogenation, and gas-phase CO oxidation. PMID:22888976

  17. Active structural growth in central Taiwan in relationship to large earthquakes and pore-fluid pressures

    NASA Astrophysics Data System (ADS)

    Yue, Li-Fan

    Central Taiwan is subject to a substantial long-term earthquake risk with a population of five million and two disastrous earthquakes in the last century, the 1935 ML=7.1 Tuntzuchiao and 1999 Mw=7.6 Chi-Chi earthquakes. Rich data from these earthquakes combined with substantial surface and subsurface data accumulated from petroleum exploration form the basis for these studies of the growth of structures in successive large earthquakes and their relationships to pore-fluid pressures. Chapter 1 documents the structural context of the bedding-parallel Chelungpu thrust that slipped in the Chi-Chi earthquake by showing for this richly instrumented earthquake the close geometric relationships between the complex 3D fault shape and the heterogeneous coseismic displacements constrained by geodesy and seismology. Chapter 2 studies the accumulation of deformation by successive large earthquakes by studying the deformation of flights of fluvial terraces deposited over the Chelungpu and adjacent Changhua thrusts, showing the deformation on a timescale of tens of thousands of years. Furthermore these two structures, involving the same stratigraphic sequence, show fundamentally different kinematics of deformation with associated contrasting hanging-wall structural geometries. The heights and shapes of deformed terraces allowed testing of existing theories of fault-related folding. Furthermore terrace dating constrains a combined shortening rate of 37 mm/yr, which is 45% of the total Taiwan plate-tectonic rate, and indicates a substantial earthquake risk for the Changhua thrust. Chapter 3 addresses the long-standing problem of the mechanics of long-thing thrust sheets, such as the Chelungpu and Changhua thrusts in western Taiwan, by presenting a natural test for the classic Hubbert-Rubey hypothesis, which argues that ambient excess pore-fluid pressure substantially reduces the effective fault friction allowing the thrusts to move. Pore-fluid pressure data obtained from 76 wells

  18. Cloxyquin (5-chloroquinolin-8-ol) is an activator of the two-pore domain potassium channel TRESK.

    PubMed

    Wright, Paul D; Weir, Gregory; Cartland, Jamie; Tickle, David; Kettleborough, Catherine; Cader, M Zameel; Jerman, Jeff

    2013-11-15

    TRESK is a two-pore domain potassium channel. Loss of function mutations have been linked to typical migraine with aura and due to TRESK’s expression pattern and role in neuronal excitability it represents a promising therapeutic target. We developed a cell based assay using baculovirus transduced U20S cells to screen for activators of TRESK. Using a thallium flux system to measure TRESK channel activity we identified Cloxyquin as a novel activator. Cloxyquin was shown to have an EC50 of 3.8 μM in the thallium assay and displayed good selectivity against other potassium channels tested. Activity was confirmed using whole cell patch electrophysiology, with Cloxyquin causing a near two fold increase in outward current. The strategy presented here will be used to screen larger compound libraries with the aim of identifying novel chemical series which may be developed into new migraine prophylactics.

  19. Cloxyquin (5-Chloroquinolin-8-ol) is an activator of the two-pore domain potassium channel TRESK.

    PubMed

    Wright, Paul D; Weir, Gregory; Cartland, Jamie; Tickle, David; Kettleborough, Catherine; Cader, Zameel; Jerman, Jeff

    2013-10-25

    TRESK is a two-pore domain potassium channel. Loss of function mutations have been linked to typical migraine with aura and due to TRESK's expression pattern and role in neuronal excitability it represents a promising therapeutic target. We developed a cell based assay using baculovirus transduced U20S cells to screen for activators of TRESK. Using a thallium flux system to measure TRESK channel activity we identified Cloxyquin as a novel activator. Cloxyquin was shown to have an EC50 of 3.8μM in the thallium assay and displayed good selectivity against other potassium channels tested. Activity was confirmed using whole cell patch electrophysiology, with Cloxyquin causing a near two fold increase in outward current. The strategy presented here will be used to screen larger compound libraries with the aim of identifying novel chemical series which may be developed into new migraine prophylactics.

  20. Active Sites Environmental Monitoring Program: Action levels

    SciTech Connect

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

    1991-10-01

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

  1. Characterization of active sites in zeolite catalysts

    SciTech Connect

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

    1997-11-01

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

  2. Structure-function studies of the muscle nicotinic acetylcholine receptor by site-directed mutagenesis in the pore region

    NASA Astrophysics Data System (ADS)

    Zhang, Haiyun

    In nicotinic acetylcholine receptors (nAChRs), as in glycine, GABA A, serotonin 5-HT3, and GluCl glutamate receptors, a leucine residue at the approximate midpoint (the 9' position) of the M2 transmembrane domain is conserved across all known subunits. We expressed the embryonic mouse muscle nAChRs with varying numbers (m* s) of subunits (2 αs, 1 β, 1 γ, and 1 δ) mutated at this position in Xenopus oocytes and discovered that mutations to serine (Leu9'Ser) result in a tenfold higher receptor sensitivity to acetylcholine (ACh) for each subunit mutated. Moreover, increases of side-chain polarity increase the sensitivity to ACh when other natural and unnatural residues are incorporated into this position. The data also indicated an especially strong interaction between the γ and δ subunits in the pore region, suggesting a specific arrangement of subunits within the pentamer. Detailed single-channel kinetic studies reveal that Leu9'Ser AChRs have (1) longer voltage- relaxation time constants, (2) longer ACh-induced openings and bursts, and (3) more frequent spontaneous openings. These effects increase with m* s. Synthesized postsynaptic currents were produced with a piezoelectric micromanipulator that delivered brief ACh pulses to multi-channel patches. Their decay time constants were, as expected, similar to the channel burst duration. Thus, both longer and more frequent openings contribute to the >=104-fold increase in the receptor sensitivity to ACh from the wild-type receptor to the receptor with m*s=4; and the highly conserved 9' leucine is crucial for the brief synaptic events that are normally observed. We also explored the effects of ligand-binding domain mutations: γD174N and δD180N (aspartic acid (D) to asparagine (N)). Macroscopic dose-response relations revealed that these mutations decrease the receptor's sensitivity to ACh. The combined effect with Leu9'Ser, however, differs from that predicted from a linear or independent sum of effects from

  3. Pore Pressure Diffusion as a possible mechanism for the Ag. Ioanis 2001 earthquake swarm activity (Gulf of Corinth, Central Greece).

    NASA Astrophysics Data System (ADS)

    Vallianatos, F.; Michas, G.; Papadakis, G.; Sammonds, P.

    2012-04-01

    The Gulf of Corinth rift (Central Greece) is one of the most seismotectonically active areas in Europe (Ambraseys and Jackson, 1990; 1997), with an important continental N-S extension of about 13 mm/yr and 6 mm/yr at the west and east part respectively (Clarke et al., 1997a). The seismicity of the area includes 5 main earthquakes of magnitude greater than 5.8 since 1960. In the western part of the rift, where the extension reaches its maximum value, earthquake swarms are often being observed (Bourouis and Cornet, 2009). Such an earthquake crisis has been occurred on 2001 at the southern margin of the west part of the rift. The crisis lasted about 100 days with a major event the Ag. Ioanis earthquake (4.3 Mw) on 8th of April 2001 (Pacchiani and Lyon-Caen, 2010). The possible relation between fluids flow and the observed earthquake swarms at the west part of the Gulf of Corinth rift has been discussed in the works of Bourouis and Cornet (2009) and Pacchiani and Lyon-Caen (2010). In the present work we examine the spatiotemporal properties of the Ag. Ioanis 2001 earthquake swarm, using data from the CRL network (http://crlab.eu/). We connect these properties to a mechanism due to pore pressure diffusion (Shapiro et al., 1997) and we estimate the hydraulic diffusivity and the permeability of the surrounding rocks. A back front of the seismicity (Parotidis et al., 2004) is also been observed, related to the migration of seismicity and the development of a quiescence region near the area of the initial pore pressure perturbation. Moreover, anisotropy of the hydraulic diffusivity has been observed, revealing the heterogeneity of the surrounding rocks and the fracture systems. This anisotropy is consistent in direction with the fault zone responsible for the Ag. Ioanis earthquake (Pacchiani and Lyon-Caen, 2010). Our results indicate that fluids flow and pore pressure perturbations are possible mechanisms for the initiation and the evolution of the Ag. Ioanis 2001

  4. The Pore Loop Domain of TRPV1 Is Required for Its Activation by the Volatile Anesthetics Chloroform and Isoflurane.

    PubMed

    Kimball, Corinna; Luo, Jialie; Yin, Shijin; Hu, Hongzhen; Dhaka, Ajay

    2015-07-01

    The environmental irritant chloroform, a naturally occurring small volatile organohalogen, briefly became the world's most popular volatile general anesthetic (VGA) before being abandoned because of its low therapeutic index. When chloroform comes in contact with skin or is ingested, it causes a painful burning sensation. The molecular basis for the pain associated with chloroform remains unknown. In this study, we assessed the role of transient receptor potential (TRP) channel family members in mediating chloroform activation and the molecular determinants of VGA activation of TRPV1. We identified the subpopulation of dorsal root ganglion (DRG) neurons that are activated by chloroform. Additionally, we transiently expressed wild-type or specifically mutated TRP channels in human embryonic kidney cells and used calcium imaging or whole-cell patch-clamp electrophysiology to assess the effects of chloroform or the VGA isoflurane on TRP channel activation. The results revealed that chloroform activates DRG neurons via TRPV1 activation. Furthermore, chloroform activates TRPV1, and it also activates TRPM8 and functions as a potent inhibitor of the noxious chemical receptor TRPA1. The results also indicate that residues in the outer pore region of TRPV1 previously thought to be required for either proton or heat activation of the channel are also required for activation by chloroform and isoflurane. In addition to identifying the molecular basis of DRG neuron activation by chloroform and the opposing effects chloroform has on different TRP channel family members, the findings of this study provide novel insights into the structural basis for the activation of TRPV1 by VGAs.

  5. Modeling Soil Pore Oxygen in Restored Wetlands

    NASA Astrophysics Data System (ADS)

    Rubol, S.; Loecke, T.; Burgin, A. J.; Franz, T.

    2015-12-01

    Soil pore oxygen (O2) is usually modeled indirectly as a function of soil moisture. However, using soil moisture to describe the oxic /anoxic status of a soil may not be sufficient accurate, especially when soil pore O2 rapidly changes, as following hydrological forcing. As first step, we use the dataset collected in the constructed wetland near Dayton, OH, by Loecke and Burgin, to reconstruct the environmental functions and re-aeration status of the soil. The dataset consist of 24 Apogee sensors and 24 soil moisture and temperature sensors located at 10 cm depth in upland, transitional and submerged zone (see Figure 1). Data were recorded over two years at temporal interval of 30 minutes. Then, we explore the capability of existing biogeochemical models to predict metabolic activity and the soil pore O2. Figure1: Restored wetland field site with soil O2 sensors (yellow stars) in upland (red), transitional (green) and submerged (blue) zones.

  6. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

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

    1985-01-01

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

  7. Characterization of differential pore-forming activities of ESAT-6 proteins from Mycobacterium tuberculosis and Mycobacterium smegmatis.

    PubMed

    Peng, Xiuli; Jiang, Guozhong; Liu, Wei; Zhang, Qi; Qian, Wei; Sun, Jianjun

    2016-02-01

    Mycobacterium tuberculosis ESAT-6 (MtbESAT-6) plays essential roles in pathogenesis. MtbESAT-6 exhibits a unique pore-forming activity (PFA) that is not found in its ortholog from non-pathogenic Mycobacterium smegmatis (MsESAT-6). Here, we characterized the differential PFAs and found that exchange of I25-H26/T25-A26 between two proteins reciprocally affected their PFAs. MtbESAT-6(IH/TA) had ~ 40% reduction, while MsESAT-6(TA/IH) fully acquired its activity similar to MtbESAT-6. Mutations of A17E, K38T, N67L or R74Q on MtbESAT-6(IH/TA) further reduced the activity, with MtbESAT-6(IH/TA-17) being the lowest. This study suggests I25-H26 as the pH-sensor essential for MsESAT-6 to fully acquire the activity, while multiple residues contributed to MtbESAT-6 PFA.

  8. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

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

    1992-02-01

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

  9. Active Sampling Device for Determining Pollutants in Surface and Pore Water – the In Situ Sampler for Biphasic Water Monitoring

    PubMed Central

    Supowit, Samuel D.; Roll, Isaac B.; Dang, Viet D.; Kroll, Kevin J.; Denslow, Nancy D.; Halden, Rolf U.

    2016-01-01

    We designed and evaluated an active sampling device, using as analytical targets a family of pesticides purported to contribute to honeybee colony collapse disorder. Simultaneous sampling of bulk water and pore water was accomplished using a low-flow, multi-channel pump to deliver water to an array of solid-phase extraction cartridges. Analytes were separated using either liquid or gas chromatography, and analysis was performed using tandem mass spectrometry (MS/MS). Achieved recoveries of fipronil and degradates in water spiked to nominal concentrations of 0.1, 1, and 10 ng/L ranged from 77 ± 12 to 110 ± 18%. Method detection limits (MDLs) were as low as 0.040–0.8 ng/L. Extraction and quantitation of total fiproles at a wastewater-receiving wetland yielded concentrations in surface water and pore water ranging from 9.9 ± 4.6 to 18.1 ± 4.6 ng/L and 9.1 ± 3.0 to 12.6 ± 2.1 ng/L, respectively. Detected concentrations were statistically indistinguishable from those determined by conventional, more laborious techniques (p > 0.2 for the three most abundant fiproles). Aside from offering time-averaged sampling capabilities for two phases simultaneously with picogram-per-liter MDLs, the novel methodology eliminates the need for water and sediment transport via in situ solid phase extraction. PMID:26905924

  10. Active Sampling Device for Determining Pollutants in Surface and Pore Water – the In Situ Sampler for Biphasic Water Monitoring

    NASA Astrophysics Data System (ADS)

    Supowit, Samuel D.; Roll, Isaac B.; Dang, Viet D.; Kroll, Kevin J.; Denslow, Nancy D.; Halden, Rolf U.

    2016-02-01

    We designed and evaluated an active sampling device, using as analytical targets a family of pesticides purported to contribute to honeybee colony collapse disorder. Simultaneous sampling of bulk water and pore water was accomplished using a low-flow, multi-channel pump to deliver water to an array of solid-phase extraction cartridges. Analytes were separated using either liquid or gas chromatography, and analysis was performed using tandem mass spectrometry (MS/MS). Achieved recoveries of fipronil and degradates in water spiked to nominal concentrations of 0.1, 1, and 10 ng/L ranged from 77 ± 12 to 110 ± 18%. Method detection limits (MDLs) were as low as 0.040–0.8 ng/L. Extraction and quantitation of total fiproles at a wastewater-receiving wetland yielded concentrations in surface water and pore water ranging from 9.9 ± 4.6 to 18.1 ± 4.6 ng/L and 9.1 ± 3.0 to 12.6 ± 2.1 ng/L, respectively. Detected concentrations were statistically indistinguishable from those determined by conventional, more laborious techniques (p > 0.2 for the three most abundant fiproles). Aside from offering time-averaged sampling capabilities for two phases simultaneously with picogram-per-liter MDLs, the novel methodology eliminates the need for water and sediment transport via in situ solid phase extraction.

  11. Active Sampling Device for Determining Pollutants in Surface and Pore Water - the In Situ Sampler for Biphasic Water Monitoring

    NASA Astrophysics Data System (ADS)

    Supowit, Samuel D.; Roll, Isaac B.; Dang, Viet D.; Kroll, Kevin J.; Denslow, Nancy D.; Halden, Rolf U.

    2016-02-01

    We designed and evaluated an active sampling device, using as analytical targets a family of pesticides purported to contribute to honeybee colony collapse disorder. Simultaneous sampling of bulk water and pore water was accomplished using a low-flow, multi-channel pump to deliver water to an array of solid-phase extraction cartridges. Analytes were separated using either liquid or gas chromatography, and analysis was performed using tandem mass spectrometry (MS/MS). Achieved recoveries of fipronil and degradates in water spiked to nominal concentrations of 0.1, 1, and 10 ng/L ranged from 77 ± 12 to 110 ± 18%. Method detection limits (MDLs) were as low as 0.040-0.8 ng/L. Extraction and quantitation of total fiproles at a wastewater-receiving wetland yielded concentrations in surface water and pore water ranging from 9.9 ± 4.6 to 18.1 ± 4.6 ng/L and 9.1 ± 3.0 to 12.6 ± 2.1 ng/L, respectively. Detected concentrations were statistically indistinguishable from those determined by conventional, more laborious techniques (p > 0.2 for the three most abundant fiproles). Aside from offering time-averaged sampling capabilities for two phases simultaneously with picogram-per-liter MDLs, the novel methodology eliminates the need for water and sediment transport via in situ solid phase extraction.

  12. Active Sampling Device for Determining Pollutants in Surface and Pore Water - the In Situ Sampler for Biphasic Water Monitoring.

    PubMed

    Supowit, Samuel D; Roll, Isaac B; Dang, Viet D; Kroll, Kevin J; Denslow, Nancy D; Halden, Rolf U

    2016-02-24

    We designed and evaluated an active sampling device, using as analytical targets a family of pesticides purported to contribute to honeybee colony collapse disorder. Simultaneous sampling of bulk water and pore water was accomplished using a low-flow, multi-channel pump to deliver water to an array of solid-phase extraction cartridges. Analytes were separated using either liquid or gas chromatography, and analysis was performed using tandem mass spectrometry (MS/MS). Achieved recoveries of fipronil and degradates in water spiked to nominal concentrations of 0.1, 1, and 10 ng/L ranged from 77 ± 12 to 110 ± 18%. Method detection limits (MDLs) were as low as 0.040-0.8 ng/L. Extraction and quantitation of total fiproles at a wastewater-receiving wetland yielded concentrations in surface water and pore water ranging from 9.9 ± 4.6 to 18.1 ± 4.6 ng/L and 9.1 ± 3.0 to 12.6 ± 2.1 ng/L, respectively. Detected concentrations were statistically indistinguishable from those determined by conventional, more laborious techniques (p > 0.2 for the three most abundant fiproles). Aside from offering time-averaged sampling capabilities for two phases simultaneously with picogram-per-liter MDLs, the novel methodology eliminates the need for water and sediment transport via in situ solid phase extraction.

  13. Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

    PubMed Central

    Raghunayakula, Sarita; Subramonian, Divya; Dasso, Mary; Kumar, Rita; Zhang, Xiang-Dong

    2015-01-01

    Annulate lamellae are cytoplasmic organelles containing stacked sheets of membranes embedded with pore complexes. These cytoplasmic pore complexes at annulate lamellae are morphologically similar to nuclear pore complexes at the nuclear envelope. Although annulate lamellae has been observed in nearly all types of cells, their biological functions are still largely unknown. Here we show that SUMO1-modification of the Ran GTPase-activating protein RanGAP1 not only target RanGAP1 to its known sites at nuclear pore complexes but also to annulate lamellae pore complexes through interactions with the Ran-binding protein RanBP2 and the SUMO-conjugating enzyme Ubc9 in mammalian cells. Furthermore, upregulation of annulate lamellae, which decreases the number of nuclear pore complexes and concurrently increases that of annulate lamellae pore complexes, causes a redistribution of nuclear transport receptors including importin α/β and the exportin CRM1 from nuclear pore complexes to annulate lamellae pore complexes and also reduces the rates of nuclear import and export. Moreover, our results reveal that importin α/β-mediated import complexes initially accumulate at annulate lamellae pore complexes upon the activation of nuclear import and subsequently disassociate for nuclear import through nuclear pore complexes in cells with upregulation of annulate lamellae. Lastly, CRM1-mediated export complexes are concentrated at both nuclear pore complexes and annulate lamellae pore complexes when the disassembly of these export complexes is inhibited by transient expression of a Ran GTPase mutant arrested in its GTP-bound form, suggesting that RanGAP1/RanBP2-activated RanGTP hydrolysis at these pore complexes is required for the dissociation of the export complexes. Hence, our findings provide a foundation for further investigation of how upregulation of annulate lamellae decreases the rates of nuclear transport and also for elucidation of the biological significance of the

  14. Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells.

    PubMed

    Raghunayakula, Sarita; Subramonian, Divya; Dasso, Mary; Kumar, Rita; Zhang, Xiang-Dong

    2015-01-01

    Annulate lamellae are cytoplasmic organelles containing stacked sheets of membranes embedded with pore complexes. These cytoplasmic pore complexes at annulate lamellae are morphologically similar to nuclear pore complexes at the nuclear envelope. Although annulate lamellae has been observed in nearly all types of cells, their biological functions are still largely unknown. Here we show that SUMO1-modification of the Ran GTPase-activating protein RanGAP1 not only target RanGAP1 to its known sites at nuclear pore complexes but also to annulate lamellae pore complexes through interactions with the Ran-binding protein RanBP2 and the SUMO-conjugating enzyme Ubc9 in mammalian cells. Furthermore, upregulation of annulate lamellae, which decreases the number of nuclear pore complexes and concurrently increases that of annulate lamellae pore complexes, causes a redistribution of nuclear transport receptors including importin α/β and the exportin CRM1 from nuclear pore complexes to annulate lamellae pore complexes and also reduces the rates of nuclear import and export. Moreover, our results reveal that importin α/β-mediated import complexes initially accumulate at annulate lamellae pore complexes upon the activation of nuclear import and subsequently disassociate for nuclear import through nuclear pore complexes in cells with upregulation of annulate lamellae. Lastly, CRM1-mediated export complexes are concentrated at both nuclear pore complexes and annulate lamellae pore complexes when the disassembly of these export complexes is inhibited by transient expression of a Ran GTPase mutant arrested in its GTP-bound form, suggesting that RanGAP1/RanBP2-activated RanGTP hydrolysis at these pore complexes is required for the dissociation of the export complexes. Hence, our findings provide a foundation for further investigation of how upregulation of annulate lamellae decreases the rates of nuclear transport and also for elucidation of the biological significance of the

  15. Incorporation and electron transfer of anthracene in pores of ZSM-5 zeolites. Effect of Brønsted acid site density.

    PubMed

    Hureau, Matthieu; Moissette, Alain; Marquis, Séverine; Brémard, Claude; Vezin, Hervé

    2009-08-01

    The sorption course of anthracene (ACENE-3) into Brønsted-acidic medium pore MFI zeolites was monitored by in situ EPR and diffuse reflectance UV-visible absorption over one year. Weighed amounts of solid ACENE-3 were merely exposed to H(n)ZSM-5 (H(n)(AlO(2))(n)(SiO(2))(96-n)), with the following Brønsted acid site (BAS) densities, n = 0.0, 0.17, 0.57, 0.95, 2.0, 3.4, 6.6, dehydrated at 623 K under argon. The weighed amounts correspond to 1 ACENE-3 per zeolite unit cell. ACENE-3 is found to be incorporated as intact molecules in purely siliceous MFI (silicalite-1). Monte Carlo simulations indicate that ACENE-3 lies in the intersection of straight and zigzag channels. In contrast, the presence of BASs on the inner surface of channels induces spontaneous ionization of ACENE-3 (ionization potential = 7.44 eV). The charge separation as ACENE-3*(+)@H(n)ZSM-5*(-) is caused by the strong Coulombic field gradient of Si-O(-)(H(+))-Al BAS in the absence of any Lewis acid site. The rate and yield of ionization are found to increase dramatically with BAS density increase. The stabilization of ACENE-3*(+)@H(n)ZSM-5*(-) is explained by the tight fit between the rod-shape ACENE-3 and the channel dimensions and especially by the compartmentalization of ejected electrons as AlO(4)H*(-) centers away from the initial site of ionization. The final charge recombination occurs after more than one year and leads to ACENE-3 occluded in the straight channel in close proximity to BAS without any protonation of ACENE-3 (pK(a) = -13.5).

  16. Design of novel 3D gene activated PEG scaffolds with ordered pore structure.

    PubMed

    Orsi, Silvia; Guarnieri, Daniela; Netti, Paolo A

    2010-03-01

    The ability to genetically modify cells seeded inside synthetic hydrogel scaffolds offers a suitable approach to induce and control tissue repair and regeneration guiding cell fate. In fact the transfected cells can act as local in vivo bioreactor, secreting plasmid encoded proteins that augment tissue regeneration processes. We have realized a DNA bioactivated high porous poly(ethylene glycol) (PEG) matrix by polyethyleneimine (PEI)/DNA complexes adsorption. As the design of the microarchitectural features of a scaffold also contributes to promote and influence cell fate, we appropriately designed the inner structure of gene activated PEG hydrogels by gelatine microparticles templating. Microarchitectural properties of the scaffold were analysed by scanning electron microscopy. 3D cell migration and transfection were monitored through time-lapse videomicroscopy and confocal laser scanning microscopy.

  17. Effects of Temperature and Pore Structure on High Surface Area-Activated Carbon Obtained from Peanut Shells.

    PubMed

    Kalpana, D; Lee, Y S

    2016-03-01

    Activated carbon was synthesized from peanut shells by treating with H3PO4 with an intention to enhance the surface area and to find its electrochemical performance in EDLC as electrode material. The powdered peanut shells were pyrolyzed at three different temperatures namely 300 degrees C, 600 degrees C and 800 degrees C respectively. The structural and surface properties of the pyrolyzed carbon materials were studied using N2 adsorption/desorption, Raman, TEM and SEM analysis. There has been remarkable increase in the surface area of the carbon pyrolyzed at 600 degrees C due to the effect of pore generations. The surface area of the 600 degrees C pyrolyzed sample was found to be 1629 m2/g. The electrochemical properties of all the samples were evaluated by cyclic voltammetry, impedance spectroscopy, and galvanostatic charge-discharge tests. The system showed excellent cycleability and a maximum specific capacitance of 291 Fg(-1) was obtained in a 0.1 M H2SO4 electrolyte solution. The effects of the various properties of the activated carbon on the EDLC performance are discussed. PMID:27455740

  18. An atypical residue in the pore of Varroa destructor GABA-activated RDL receptors affects picrotoxin block and thymol modulation

    PubMed Central

    Price, Kerry L.; Lummis, Sarah C.R.

    2014-01-01

    GABA-activated RDL receptors are the insect equivalent of mammalian GABAA receptors, and play a vital role in neurotransmission and insecticide action. Here we clone the pore lining M2 region of the Varroa mite RDL receptor and show that it has 4 atypical residues when compared to M2 regions of most other insects, including bees, which are the major host of Varroa mites. We create mutant Drosophila RDL receptors containing these substitutions and characterise their effects on function. Using two electrode voltage clamp electrophysiology we show that one substitution (T6′M) ablates picrotoxin inhibition and increases the potency of GABA. This mutation also alters the effect of thymol, which enhances both insect and mammalian GABA responses, and is widely used as a miticide. Thymol decreases the GABA EC50 of WT receptors, enhancing responses, but in T6′M-containing receptors it is inhibitory. The other 3 atypical residues have no major effects on either the GABA EC50, the picrotoxin potency or the effect of thymol. In conclusion we show that the RDL 6′ residue is important for channel block, activation and modulation, and understanding its function also has the potential to prove useful in the design of Varroa-specific insecticidal agents. PMID:25460510

  19. The M34A mutant of Connexin26 reveals active conductance states in pore-suspending membranes

    PubMed Central

    Gaßmann, Oliver; Kreir, Mohamed; Ambrosi, Cinzia; Pranskevich, Jennifer; Oshima, Atsunori; Röling, Christian; Sosinsky, Gina; Fertig, Niels; Steinem, Claudia

    2009-01-01

    Connexin26 (Cx26) is a member of the connexin family, the building blocks for gap junction intercellular channels. These dodecameric assemblies are involved in gap junction-mediated cell-cell communication allowing the passage of ions and small molecules between two neighboring cells. Mutations in Cx26 lead to the disruption of gap junction-mediated intercellular communication with consequences such as hearing loss and skin disorders. We show here that a mutant of Cx26, M34A, forms an active hemichannel in lipid bilayer experiments. A comparison with the Cx26 wild-type is presented. Two different techniques using micro/nano-structured substrates for the formation of pore-suspending lipid membranes are used. We reconstituted the Cx26 wild-type and Cx26M34A into artificial lipid bilayers and observed single channel activity for each technique, with conductance levels of around 35, 70 and 165 pS for the wildtype. The conductance levels of Cx26M34A were found at around 45 and 70 pS. PMID:19236918

  20. Effects of Temperature and Pore Structure on High Surface Area-Activated Carbon Obtained from Peanut Shells.

    PubMed

    Kalpana, D; Lee, Y S

    2016-03-01

    Activated carbon was synthesized from peanut shells by treating with H3PO4 with an intention to enhance the surface area and to find its electrochemical performance in EDLC as electrode material. The powdered peanut shells were pyrolyzed at three different temperatures namely 300 degrees C, 600 degrees C and 800 degrees C respectively. The structural and surface properties of the pyrolyzed carbon materials were studied using N2 adsorption/desorption, Raman, TEM and SEM analysis. There has been remarkable increase in the surface area of the carbon pyrolyzed at 600 degrees C due to the effect of pore generations. The surface area of the 600 degrees C pyrolyzed sample was found to be 1629 m2/g. The electrochemical properties of all the samples were evaluated by cyclic voltammetry, impedance spectroscopy, and galvanostatic charge-discharge tests. The system showed excellent cycleability and a maximum specific capacitance of 291 Fg(-1) was obtained in a 0.1 M H2SO4 electrolyte solution. The effects of the various properties of the activated carbon on the EDLC performance are discussed.

  1. Altered RNA processing and export lead to retention of mRNAs near transcription sites and nuclear pore complexes or within the nucleolus.

    PubMed

    Paul, Biplab; Montpetit, Ben

    2016-09-01

    Many protein factors are required for mRNA biogenesis and nuclear export, which are central to the eukaryotic gene expression program. It is unclear, however, whether all factors have been identified. Here we report on a screen of >1000 essential gene mutants in Saccharomyces cerevisiae for defects in mRNA processing and export, identifying 26 mutants with defects in this process. Single-molecule FISH data showed that the majority of these mutants accumulated mRNA within specific regions of the nucleus, which included 1) mRNAs within the nucleolus when nucleocytoplasmic transport, rRNA biogenesis, or RNA processing and surveillance was disrupted, 2) the buildup of mRNAs near transcription sites in 3'-end processing and chromosome segregation mutants, and 3) transcripts being enriched near nuclear pore complexes when components of the mRNA export machinery were mutated. These data show that alterations to various nuclear processes lead to the retention of mRNAs at discrete locations within the nucleus.

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

    SciTech Connect

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

    1993-12-01

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

  3. Triggered pore-forming agents

    DOEpatents

    Bayley, Hagan; Walker, Barbara J.; Chang, Chung-yu; Niblack, Brett; Panchal, Rekha

    1998-01-01

    An inactive pore-forming agent which is activated to lytic function by a condition such as pH, light, heat, reducing potential, or metal ion concentration, or substance such as a protease, at the surface of a cell.

  4. Connecting Wastewater Injection and Seismicity through Pore Pressure

    NASA Astrophysics Data System (ADS)

    Ge, S.; Weingarten, M.; Person, M. A.; Bekins, B. A.

    2014-12-01

    Increased seismicity in recent years in some geologically quiescent regions in the US has been linked to wastewater injection associated with oil and gas production. While seismicity in some cases appears to be well correlated with injection activities, in many other injection locations no seismicity has been reported. How pore pressures generated from injection propagate spatially and evolve temporally is likely a key control in inducing earthquakes, providing a physical linkage between injection activity and seismicity occurrence. Yet, the linkage remains controversial and inconclusive, in spite of the basic physics of pore pressure propagation being well established. This study aims at better understanding the physical processes of pore pressure propagation around injection sites and identifying factors that are most likely contributors to induced seismicity. Numerical modeling suggests that pore pressure increases in the Jones seismicity swarm northeast of Oklahoma City were primarily from several high rate injection wells. Preliminary analysis on injection and seismicity data from Greeley, Colorado also points to a potential pore pressure link between high injection rates and seismicity. Modeling of pore pressures in the Lake County, Ohio, illustrates that permeable faults in the crystalline basement could facilitate pore pressure propagation from injection in the basal aquifer and host earthquakes, which could explain the earthquakes that occurred in the mid-1980s. In many of the above examples, wastewater injection in basal aquifers promoted downward propagation of pore pressures into the crystalline basement. In connecting injection and seismicity through pore pressure propagation, high rate injection wells and permeable basement faults are merging as important players contributing to induced seismicity. It is the intention of this study that findings like these would provide a scientific basis to inform future regulations and policies on wastewater

  5. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

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

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

  6. Poring over two-pore channel pore mutants

    PubMed Central

    Penny, Christopher J.; Patel, Sandip

    2016-01-01

    Two-pore channels are members of the voltage-gated ion channel superfamily. They localise to the endolysosomal system and are likely targets for the Ca2+ mobilising messenger NAADP. In this brief review, we relate mutagenesis of the TPC pore to a recently published homology model and discuss how pore mutants are informing us of TPC function. Molecular physiology of these ubiquitous proteins is thus emerging. PMID:27226934

  7. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  8. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

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

  11. Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels.

    PubMed

    Peters, Christian J; Yu, Haibo; Tien, Jason; Jan, Yuh Nung; Li, Min; Jan, Lily Yeh

    2015-03-17

    TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative pore-lining basic residues significantly altered the IC50 of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore.

  12. Designed membrane channels and pores.

    PubMed

    Bayley, H

    1999-02-01

    Advances in the synthesis and assembly of designed membrane channels and pores include addressable template-assisted synthetic protein (TASP) syntheses of helix bundles, the production of a new class of nanotubes and the ability to purify hetero-oligomeric pores. Channels and pores with altered functional properties and with built-in triggers and switches have been prepared. Progress in applications has been greatest in sensor technology, where sensor elements based on ligand activation, channel selectivity and channel block have been made. Structural information about natural membrane proteins is emerging to inspire new designs.

  13. Typology, morphology and connectivity of pore space in claystones from reference site for research using BIB, FIB and cryo-SEM methods

    NASA Astrophysics Data System (ADS)

    Desbois, G.; Urai, J. L.; Houben, M. E.; Sholokhova, Y.

    2010-06-01

    Detailed investigation of the morphology of the pore space in clay is a key factor in understanding the sealing capacity, coupled flows, capillary processes and associated deformation present in mudstones. Actually, the combination of ion milling tools (FIB and BIB), cryogenic techniques and SEM imaging offers a new alternative to study in-situ elusive microstructures in wet geomaterials and has the high potential to make a step change in our understanding of how fluids occur in pore space. By using this range of techniques, it is possible to quantify porosity, stabilize in-situ fluids in pore space, preserve the natural structures at nm-scale, produce high quality polished cross-sections for high resolution SEM imaging and reconstruct accurately microstructure networks in 3D by serial cross sectioning.

  14. Impacts of simulated drought on pore water chemistry of peatlands.

    PubMed

    Juckers, Myra; Watmough, Shaun A

    2014-01-01

    Northern peatlands are increasingly threatened by climate change and industrial activities. This study examined the impact of simulated droughts on pore water chemistry at six peatlands in Sudbury, Ontario, that differ in copper (Cu), nickel (Ni) and cobalt (Co) contamination, including a site that had been previously limed. All sites responded similarly to simulated drought: pore water pH declined significantly following the 30 day drought and the decline was greater following the 60 day drought treatment. The decline in pore water pH was due to increasing sulphate concentrations, whereas nitrate increased more in the 60 day drought treatment. Decreases in pH were accompanied by large increases in Ni and Co that greatly exceeded provincial water quality guidelines. In contrast, dissolved organic carbon (DOC) concentrations decreased significantly following drought, along with concentrations of Cu and Al, which are strongly complexed by organic acids.

  15. 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.

  16. Effects of pore sizes and oxygen-containing functional groups on desulfurization activity of Fe/NAC prepared by ultrasonic-assisted impregnation

    NASA Astrophysics Data System (ADS)

    Shu, Song; Guo, Jia-Xiu; Liu, Xiao-Li; Wang, Xue-Jiao; Yin, Hua-Qiang; Luo, De-Ming

    2016-01-01

    A series of Fe-loaded activated carbons treated by HNO3 (Fe/NAC) were prepared by incipient impregnation method with or without ultrasonic assistance and characterized using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy with energy disperse spectroscope (SEM-EDS), transmission electron microscopy (TEM) and N2 adsorption/desorption. The desulfurization activities were evaluated at a fixed bed reactor under a mixed gas simulated from flue gas. The results showed that desulfurization activity from excellent to poor is as follows: Fe/NAC-60 > Fe/NAC-80 > Fe/NAC-30 > Fe/NAC-15 > Fe/NAC-0 > Fe/NAC-100 > NAC. Fe/NAC-60 exhibits the best desulfurization activity and has breakthrough sulfur capacity of 319 mg/g and breakthrough time of 540 min. The introduction of ultrasonic oscillation does not change the form of Fe oxides on activated carbon but can change the dispersion and relative contents of Fe3O4. The types of oxygen-containing functional groups have no obvious change for all samples but the texture properties show some differences when they are oscillated for different times. The fresh Fe/NAC-60 has a surface area of 1045 m2/g and total pore volume of 0.961 cm3/g with micropore volume of 0.437 cm3/g and is larger than Fe/NAC-0 (823 m2/g, 0.733 and 0.342 cm3/g). After desulfurization, surface area and pore volume of all samples decrease significantly, and those of the exhausted Fe/NAC-60 decrease to 233 m2/g and 0.481 cm3/g, indicating that some byproducts deposit on surface to cover pores. Pore size distribution influences SO2 adsorption, and fresh Fe/NAC-60 has more pore widths centralized at about 0.7 nm and 1.0-2.0 nm and corresponds to an excellent desulfurization activity, showing that micropore is conducive to the removal of SO2.

  17. 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.

  18. 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.

  19. 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.

  20. The influence of pore size and surface area of activated carbons on the performance of ionic liquid based supercapacitors.

    PubMed

    Pohlmann, Sebastian; Lobato, Belén; Centeno, Teresa A; Balducci, Andrea

    2013-10-28

    This study analyses and compares the behaviour of 5 commercial porous carbons in the ionic liquid N-butyl-N-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide (PYR14TFSI) and its mixture with propylene carbonate (PC) as electrolytes. The results of this investigation show that the existence of a distribution of pore sizes and/or constrictions at the entrance of the pores leads to significant changes in the specific capacitance of the investigated materials. The use of PYR14TFSI as an electrolyte has a positive effect on the EDLC energy storage, but its high viscosity limits the power density. The mixture 50 : 50 wt% propylene carbonate-PYR14TFSI provides high operative voltage as well as low viscosity and thus notably enhances EDLC operation. PMID:24019082

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

    PubMed Central

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

    1997-01-01

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

  2. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

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

  3. Polystyrene-Divinylbenzene-Based Adsorbents Reduce Endothelial Activation and Monocyte Adhesion Under Septic Conditions in a Pore Size-Dependent Manner.

    PubMed

    Eichhorn, Tanja; Rauscher, Sabine; Hammer, Caroline; Gröger, Marion; Fischer, Michael B; Weber, Viktoria

    2016-10-01

    Endothelial activation with excessive recruitment and adhesion of immune cells plays a central role in the progression of sepsis. We established a microfluidic system to study the activation of human umbilical vein endothelial cells by conditioned medium containing plasma from lipopolysaccharide-stimulated whole blood or from septic blood and to investigate the effect of adsorption of inflammatory mediators on endothelial activation. Treatment of stimulated whole blood with polystyrene-divinylbenzene-based cytokine adsorbents (average pore sizes 15 or 30 nm) prior to passage over the endothelial layer resulted in significantly reduced endothelial cytokine and chemokine release, plasminogen activator inhibitor-1 secretion, adhesion molecule expression, and in diminished monocyte adhesion. Plasma samples from sepsis patients differed substantially in their potential to induce endothelial activation and monocyte adhesion despite their almost identical interleukin-6 and tumor necrosis factor-alpha levels. Pre-incubation of the plasma samples with a polystyrene-divinylbenzene-based adsorbent (30 nm average pore size) reduced endothelial intercellular adhesion molecule-1 expression to baseline levels, resulting in significantly diminished monocyte adhesion. Our data support the potential of porous polystyrene-divinylbenzene-based adsorbents to reduce endothelial activation under septic conditions by depletion of a broad range of inflammatory mediators. PMID:27503310

  4. Polystyrene-Divinylbenzene-Based Adsorbents Reduce Endothelial Activation and Monocyte Adhesion Under Septic Conditions in a Pore Size-Dependent Manner.

    PubMed

    Eichhorn, Tanja; Rauscher, Sabine; Hammer, Caroline; Gröger, Marion; Fischer, Michael B; Weber, Viktoria

    2016-10-01

    Endothelial activation with excessive recruitment and adhesion of immune cells plays a central role in the progression of sepsis. We established a microfluidic system to study the activation of human umbilical vein endothelial cells by conditioned medium containing plasma from lipopolysaccharide-stimulated whole blood or from septic blood and to investigate the effect of adsorption of inflammatory mediators on endothelial activation. Treatment of stimulated whole blood with polystyrene-divinylbenzene-based cytokine adsorbents (average pore sizes 15 or 30 nm) prior to passage over the endothelial layer resulted in significantly reduced endothelial cytokine and chemokine release, plasminogen activator inhibitor-1 secretion, adhesion molecule expression, and in diminished monocyte adhesion. Plasma samples from sepsis patients differed substantially in their potential to induce endothelial activation and monocyte adhesion despite their almost identical interleukin-6 and tumor necrosis factor-alpha levels. Pre-incubation of the plasma samples with a polystyrene-divinylbenzene-based adsorbent (30 nm average pore size) reduced endothelial intercellular adhesion molecule-1 expression to baseline levels, resulting in significantly diminished monocyte adhesion. Our data support the potential of porous polystyrene-divinylbenzene-based adsorbents to reduce endothelial activation under septic conditions by depletion of a broad range of inflammatory mediators.

  5. A Micelle Fusion-Aggregation Assembly Approach to Mesoporous Carbon Materials with Rich Active Sites for Ultrasensitive Ammonia Sensing.

    PubMed

    Luo, Wei; Zhao, Tao; Li, Yuhui; Wei, Jing; Xu, Pengcheng; Li, Xinxin; Wang, Youwei; Zhang, Wenqing; Elzatahry, Ahmed A; Alghamdi, Abdulaziz; Deng, Yonghui; Wang, Lianjun; Jiang, Wan; Liu, Yong; Kong, Biao; Zhao, Dongyuan

    2016-09-28

    Nanostructured carbon materials have received considerable attention due to their special physicochemical properties. Herein, ordered mesoporous carbons (OMCs) with two-dimension (2D) hexagonal mesostructure and unique buckled large mesopores have successfully been synthesized via a micelle fusion-aggregation assembly method by using poly(ethylene oxide)-block-polystyrene (PEO-b-PS) diblock copolymers as a template and resorcinol-based phenolic resin as a carbon precursor. The obtained ordered mesoporous carbons possess unique fiber-like morphology, specific surface area of 571-880 m(2)/g, pore volume of 0.54 cm(3)/g and large mesopores (up to 36.3 nm) and high density of active sites (i.e., carboxylic groups) of 0.188/nm(2). Gas sensor based on the ordered mesoporous carbons exhibits an excellent performance in sensing NH3 at a low temperature with fast response (<2 min), ultralow limit of detection (<1 ppm), and good selectivity, due to the large pore sizes, high surface area and rich active sites in the carbon pore walls.

  6. The pore space scramble

    NASA Astrophysics Data System (ADS)

    Gormally, Alexandra; Bentham, Michelle; Vermeylen, Saskia; Markusson, Nils

    2015-04-01

    Climate change and energy security continue to be the context of the transition to a secure, affordable and low carbon energy future, both in the UK and beyond. This is reflected in for example, binding climate policy targets at the EU level, the introduction of renewable energy targets, and has also led to an increasing interest in Carbon Capture and Storage (CCS) technology with its potential to help mitigate against the effects of CO2 emissions from fossil fuel burning. The UK has proposed a three phase strategy to integrate CCS into its energy system in the long term focussing on off-shore subsurface storage (DECC, 2014). The potential of CCS therefore, raises a number of challenging questions and issues surrounding the long-term storage of CO2 captured and injected into underground spaces and, alongside other novel uses of the subsurface, contributes to opening a new field for discussion on the governance of the subsurface. Such 'novel' uses of the subsurface have lead to it becoming an increasingly contested space in terms of its governance, with issues emerging around the role of ownership, liability and property rights of subsurface pore space. For instance, questions over the legal ownership of pore space have arisen with ambiguity over the legal standpoint of the surface owner and those wanting to utilise the pore space for gas storage, and suggestions of whether there are depths at which legal 'ownership' becomes obsolete (Barton, 2014). Here we propose to discuss this 'pore space scramble' and provide examples of the competing trajectories of different stakeholders, particularly in the off-shore context given its priority in the UK. We also propose to highlight the current ambiguity around property law of pore space in the UK with reference to approaches currently taken in different national contexts. Ultimately we delineate contrasting models of governance to illustrate the choices we face and consider the ethics of these models for the common good

  7. Soils, Pores, and NMR

    NASA Astrophysics Data System (ADS)

    Pohlmeier, Andreas; Haber-Pohlmeier, Sabina; Haber, Agnes; Sucre, Oscar; Stingaciu, Laura; Stapf, Siegfried; Blümich, Bernhard

    2010-05-01

    Within Cluster A, Partial Project A1, the pore space exploration by means of Nuclear Magnetic Resonance (NMR) plays a central role. NMR is especially convenient since it probes directly the state and dynamics of the substance of interest: water. First, NMR is applied as relaxometry, where the degree of saturation but also the pore geometry controls the NMR signature of natural porous systems. Examples are presented where soil samples from the Selhausen, Merzenhausen (silt loams), and Kaldenkirchen (sandy loam) test sites are investigated by means of Fast Field Cycling Relaxometry at different degrees of saturation. From the change of the relaxation time distributions with decreasing water content and by comparison with conventional water retention curves we conclude that the fraction of immobile water is characterized by T1 < 5 ms. Moreover, the dependence of the relaxation rate on magnetic field strength allows the identification of 2D diffusion at the interfaces as the mechanism which governs the relaxation process (Pohlmeier et al. 2009). T2 relaxation curves are frequently measured for the rapid characterization of soils by means of the CPMG echo train. Basically, they contain the same information about the pore systems like T1 curves, since mostly the overall relaxation is dominated by surface relaxivity and the surface/volume ratio of the pores. However, one must be aware that T2 relaxation is additionally affected by diffusion in internal gradients, and this can be overcome by using sufficiently short echo times and low magnetic fields (Stingaciu et al. 2009). Second, the logic continuation of conventional relaxation measurements is the 2-dimensional experiment, where prior to the final detection of the CPMG echo train an encoding period is applied. This can be T1-encoding by an inversion pulse, or T2 encoding by a sequence of 90 and 180° pulses. During the following evolution time the separately encoded signals can mix and this reveals information about

  8. The chimeric approach reveals that differences in the TRPV1 pore domain determine species-specific sensitivity to block of heat activation.

    PubMed

    Papakosta, Marianthi; Dalle, Carine; Haythornthwaite, Alison; Cao, Lishuang; Stevens, Edward B; Burgess, Gillian; Russell, Rachel; Cox, Peter J; Phillips, Stephen C; Grimm, Christian

    2011-11-11

    The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.

  9. The Chimeric Approach Reveals That Differences in the TRPV1 Pore Domain Determine Species-specific Sensitivity to Block of Heat Activation*

    PubMed Central

    Papakosta, Marianthi; Dalle, Carine; Haythornthwaite, Alison; Cao, Lishuang; Stevens, Edward B.; Burgess, Gillian; Russell, Rachel; Cox, Peter J.; Phillips, Stephen C.; Grimm, Christian

    2011-01-01

    The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa. PMID:21911503

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

    SciTech Connect

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

    1986-03-20

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

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

    PubMed

    Chou, Kuo-Chen; Cai, Yu-dong

    2004-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2006-11-01

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

  13. A small ribozyme with dual-site kinase activity

    PubMed Central

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

    2012-01-01

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

  14. PROTECTIVE EFFECTS OF POTASSIUM TRANSPORT IN MITOCHONDRIA FROM RAT MYOMETRIUM UNDER ACTIVATION OF MITOCHONDRIAL PERMEABILITY TRANSITION PORE.

    PubMed

    Vadzyuk, O B

    2015-01-01

    We demonstrated using PBFI K(+)-sensitive fluorescent probe an enhancement of both components of K(+)-cycle--the ATP-sensitive K(+)-uptake and quinine-sensitive K+/H(+)-exchange--under the Ca2+ induced opening of mitochondrial permeability transition pore (MPTP) in rat myometrium mitochondria. Addition of CaCl2 (100 μM to K(+)-free medium results in the enhancement of reactive oxygen species (ROS) production, which was eliminated by cyclosporine A. Addition of CaCl2 to K(+)-rich medium did not increase the rate of ROS production, but blocking of mitoK+(ATP)-channels with glybenclamide (10 mcM increased production of ROS. We conclude that K(+)-cycle exerts a protective influence in mitochondria from rat myometrium by regulation of matrix volume and rate of ROS production under the condition of Ca(2+)-induced MPTP.

  15. [Structure and function of pore-forming proteins from bacteria of the genus Yersinia: I. Isolation and a comparison of physicochemical properties and functional activity of Yersinia porins].

    PubMed

    Vostrikova, O P; Kim, N Iu; Likhatskaia, G N; Guzev, K V; Vakorina, T I; Khomenko, V A; Novikova, O D; Solov'eva, T F

    2006-01-01

    The molecular organization and functional activity of porins isolated from the outer membrane (OM) of the Yersinia enterocolitica and three phylogenetically close nonpathogenic Yersinia species (Y. intermedia, Y. kristensenii, and Y. frederiksenii) cultured at 6-8 degrees C were comparatively studied for the first time. The proteins were isolated in two molecular forms (trimeric and monomeric), and their spatial structures were characterized by the methods of optical spectroscopy, CD and intrinsic protein fluorescence. The studied porins were shown to belong to the beta-structural proteins (they have 59-96% total beta structures and 0-17% alpha helices). The spatial structures of the proteins were demonstrated to depend on the nature of the detergent used for solubilization. Unlike the enterobacterial pore-forming proteins, the porin trimers are less stable to sodium dodecyl sulfate (SDS). The spatial structures of the porins become more compact after the substitution of octyl beta-D-glucoside for SDS: the content of beta structures increases and the accessibility of Trp residues to solvent decreases. It was established with the use of the technique of bilayer lipid membranes that the functional properties of the porins are similar to those of the OmpF proteins of Gram-negative bacteria. Trimers are functionally active forms of the porins. Special features of the pore-forming activity of the Yersinia porins were revealed to depend on the microorganism species and the value of the membrane potential.

  16. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

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

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

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

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

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

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

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

  19. Modeling Activity of Very-Low-Frequency Earthquakes in Shallow Subduction Zone Considering Splay Faults and High Pore Pressure Zones

    NASA Astrophysics Data System (ADS)

    Shibazaki, B.; Ito, Y.; Ujiie, K.

    2010-12-01

    Recent observations reveal that very-low-frequency (VLF) earthquakes occur in the shallow subduction zones in the Nankai trough, Hyuganada, and off the coast of Tokachi, Japan (Obara and Ito, 2005; Asano et al., 2008; Obana and Kodaira, 2009). The ongoing super drilling project, Nankai Trough Seismogenic Zone Experiment (NanTroSEIZE), involves sampling the core of seismogenic faults and conducting analyses, experiments, and in-situ borehole measurements at the Nankai trough where VLF earthquakes occur. The data obtained in this project will be used to develop a model of VLF earthquakes that integrates seismological observations, laboratory experimental results, and geological observations. In the present study, first, we perform 2D quasi-dynamic modeling of VLF earthquakes in an elastic half-space on the basis of a rate- and state-dependent friction law. We set a local unstable zone in a shallow stable zone. To explain very low stress drops and short recurrence intervals of VLF earthquakes, the effective stress is assumed to be around 0.2 MPa. The results indicate that VLF earthquakes are unstable slips that occur under high pore pressure conditions. The probable causes for the high pore pressure along the faults of VLF earthquakes are the sediment compaction and dehydration that occur during smectite-to-illite transition in the shallow subduction zone. Then, we model the generation process of VLF earthquakes by considering splay faults and the occurrences of large subduction earthquakes. We set the local unstable zones with high pore pressure in the stable splay fault zones. We assume the long-term average slip velocity of the splay faults, and that the shear stress is accumulated by the delay of the fault slip from the long-term slip motion. Depending on the frictional properties of the shallow splay faults, two types of VLF earthquakes can occur. When the effective stress is low all over the splay faults, the rupture of large earthquakes propagates to the

  20. Probing Binding Sites and Mechanisms of Action of an IKs Activator by Computations and Experiments

    PubMed Central

    Xu, Yu; Wang, Yuhong; Zhang, Mei; Jiang, Min; Rosenhouse-Dantsker, Avia; Wassenaar, Tsjerk; Tseng, Gea-Ny

    2015-01-01

    The slow delayed rectifier (IKs) channel is composed of the KCNQ1 channel and KCNE1 auxiliary subunit, and functions to repolarize action potentials in the human heart. IKs activators may provide therapeutic efficacy for treating long QT syndromes. Here, we show that a new KCNQ1 activator, ML277, can enhance IKs amplitude in adult guinea pig and canine ventricular myocytes. We probe its binding site and mechanism of action by computational analysis based on our recently reported KCNQ1 and KCNQ1/KCNE1 3D models, followed by experimental validation. Results from a pocket analysis and docking exercise suggest that ML277 binds to a side pocket in KCNQ1 and the KCNE1-free side pocket of KCNQ1/KCNE1. Molecular-dynamics (MD) simulations based on the most favorable channel/ML277 docking configurations reveal a well-defined ML277 binding space surrounded by the S2-S3 loop and S4-S5 helix on the intracellular side, and by S4–S6 transmembrane helices on the lateral sides. A detailed analysis of MD trajectories suggests two mechanisms of ML277 action. First, ML277 restricts the conformational dynamics of the KCNQ1 pore, optimizing K+ ion coordination in the selectivity filter and increasing current amplitudes. Second, ML277 binding induces global motions in the channel, including regions critical for KCNQ1 gating transitions. We conclude that ML277 activates IKs by binding to an intersubunit space and allosterically influencing pore conductance and gating transitions. KCNE1 association protects KCNQ1 from an arrhythmogenic (constitutive current-inducing) effect of ML277, but does not preclude its current-enhancing effect. PMID:25564853

  1. Potassium channels as multi-ion single-file pores

    PubMed Central

    1978-01-01

    A literature review reveals many lines of evidence that both delayed rectifier and inward rectifier potassium channels are multi-ion pores. These include unidirectional flux ratios given by the 2--2.5 power of the electrochemical activity ratio, very steeply voltage-dependent block with monovalent blocking ions, relief of block by permeant ions added to the side opposite from the blocking ion, rectification depending on E--EK, and a minimum in the reversal potential or conductance as external K+ ions are replaced by an equivalent concentration of T1+ ions. We consider a channel with a linear sequence of energy barriers and binding sites. The channel can be occupied by more than one ion at a time, and ions hop in single file into vacant sites with rate constants that depend on barrier heights, membrane potential, and interionic repulsion. Such multi-ion models reproduce qualitatively the special flux properties of potassium channels when the barriers for hopping out of the pore are larger than for hopping between sites within the pore and when there is repulsion between ions. These conditions also produce multiple maxima in the conductance-ion activity relationship. In agreement with Armstrong's hypothesis (1969. J. Gen. Physiol. 54:553--575), inward rectification may be understood in terms of block by an internal blocking cation. Potassium channels must have at least three sites and often contain at least two ions at a time. PMID:722275

  2. Luminescent microporous metal-organic framework with functional Lewis basic sites on the pore surface: Quantifiable evaluation of luminescent sensing mechanisms towards Fe3+

    NASA Astrophysics Data System (ADS)

    Jin, Jun-Cheng; Guo, Rui-Li; Zhang, Wen-Yan; Jiang, Chen; Wang, Yao-Yu

    2016-11-01

    A systematic study has been conducted on a novel luminescent metal-organic framework, {[Zn(bpyp)(L-OH)]·DMF·2H2O}n (1), to explore its sensing mechanisms to Fe3+. Structure analyses show that compound 1 exist pyridine N atoms and -OH groups on the pore surface for specific sensing of metal ions via Lewis acid-base interactions. On this consideration, the quenching mechanisms are studied and the processes are controlled by multiple mechanisms in which dynamic and static mechanisms are calculated, achieving the quantification evaluation of the quenching process. This work not only achieves the quantitative evaluation of the luminescence quenching but also provides certain insights into the quenching process, and the possible mechanisms explored in this work may inspire future research and design of target luminescent metal-organic frameworks (LMOFs) with specific functions.

  3. Triggered pore-forming agents

    DOEpatents

    Bayley, H.; Walker, B.J.; Chang, C.Y.; Niblack, B.; Panchal, R.

    1998-07-07

    An inactive pore-forming agent is revealed which is activated to lytic function by a condition such as pH, light, heat, reducing potential, or metal ion concentration, or substance such as a protease, at the surface of a cell. 30 figs.

  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 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.

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

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

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

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

    PubMed Central

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

    1989-01-01

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

  8. FABRY-PEROT VERSUS SLIT SPECTROPOLARIMETRY OF PORES AND ACTIVE NETWORK: ANALYSIS OF IBIS AND HINODE DATA

    SciTech Connect

    Judge, Philip G.; De Wijn, Alfred; Tritschler, Alexandra; Uitenbroek, Han

    2010-02-20

    We discuss spectropolarimetric measurements of photospheric (Fe I 630.25 nm) and chromospheric (Ca II 854.21 nm) spectral lines in and around small magnetic flux concentrations, including a pore. Our long-term goal is to diagnose properties of the magnetic field near the base of the corona. We compare ground-based two-dimensional spectropolarimetric measurem ents with (almost) simultaneous space-based slit spectropolarimetry. We address the question of noise and crosstalk in the measurements and attempt to determine the suitability of Ca II measurements with imaging spectropolarimeters for the determination of chromospheric magnetic fields. The ground-based observations were obtained 2008 May 20, with the Interferometric Bidimensional Spectrometer (IBIS) in spectropolarimetric mode operated at the Dunn Solar Telescope at Sunspot, NM. The space observations were obtained with the Spectro-Polarimeter of the Solar Optical Telescope aboard the Japanese Hinode satellite. The agreement between the near-simultaneous co-spatial IBIS and Hinode Stokes-V profiles at 630.25 nm is excellent, with V/I amplitudes compatible to within 1%. The IBIS QU measurements are affected by residual crosstalk from V, arising from calibration inaccuracies, not from any inherent limitation of imaging spectroscopy. We use a Principal Component Analysis to quantify the detected crosstalk. QU profiles with V crosstalk subtracted are in good agreement with the Hinode measurements, but are noisier owing to fewer collected photons. Chromospheric magnetic fields are notoriously difficult to constrain by polarization of Ca II lines alone. However, we demonstrate that high cadence, high angular resolution monochromatic images of fibrils in Ca II and Halpha, seen clearly in IBIS observations, can be used to improve the magnetic field constraints, under conditions of high electrical conductivity. Such work is possible only with time series data sets from two-dimensional spectroscopic instruments such as

  9. Linking biofilm growth to fouling and aeration performance of fine-pore diffuser in activated sludge.

    PubMed

    Garrido-Baserba, Manel; Asvapathanagul, Pitiporn; McCarthy, Graham W; Gocke, Thomas E; Olson, Betty H; Park, Hee-Deung; Al-Omari, Ahmed; Murthy, Sudhir; Bott, Charles B; Wett, Bernhard; Smeraldi, Joshua D; Shaw, Andrew R; Rosso, Diego

    2016-03-01

    Aeration is commonly identified as the largest contributor to process energy needs in the treatment of wastewater and therefore garners significant focus in reducing energy use. Fine-pore diffusers are the most common aeration system in municipal wastewater treatment. These diffusers are subject to fouling and scaling, resulting in loss in transfer efficiency as biofilms form and change material properties producing larger bubbles, hindering mass transfer and contributing to increased plant energy costs. This research establishes a direct correlation and apparent mechanistic link between biofilm DNA concentration and reduced aeration efficiency caused by biofilm fouling. Although the connection between biofilm growth and fouling has been implicit in discussions of diffuser fouling for many years, this research provides measured quantitative connection between the extent of biofouling and reduced diffuser efficiency. This was clearly established by studying systematically the deterioration of aeration diffusers efficiency during a 1.5 year period, concurrently with the microbiological study of the biofilm fouling in order to understand the major factors contributing to diffuser fouling. The six different diffuser technologies analyzed in this paper included four different materials which were ethylene-propylene-diene monomer (EPDM), polyurethane, silicone and ceramic. While all diffusers foul eventually, some novel materials exhibited fouling resistance. The material type played a major role in determining the biofilm characteristics (i.e., growth rate, composition, and microbial density) which directly affected the rate and intensity at what the diffusers were fouled, whereas diffuser geometry exerted little influence. Overall, a high correlation between the increase in biofilm DNA and the decrease in αF was evident (CV < 14.0 ± 2.0%). By linking bacterial growth with aeration efficiency, the research was able to show quantitatively the causal connection

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

    SciTech Connect

    Kefang Jiao; Song Li; Zhengzheng Lu

    1996-12-31

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

  11. 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.

  12. Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

    PubMed Central

    Neely, Alan; Hidalgo, Patricia

    2014-01-01

    Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

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

    PubMed Central

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

    2008-01-01

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

  14. Factors Affecting the Sealing Efficiency of Low-k Dielectric Surface Pores Using Successive He and Ar/NH3 Plasma Treatment

    NASA Astrophysics Data System (ADS)

    Shoeb, Juline; Kushner, Mark

    2009-10-01

    Sequential treatment of porous SiCOH by He and NH3 plasmas is effective at sealing pores while maintaining the low-k of the dielectric. He plasmas activate surface sites to accelerate the reactions responsible for pore sealing. Additional NH3 plasma treatment completes the sealing through formation of Si-N, C-N and N-N bonds resulting from the adsorption of NHx. To seal pores, sufficient He plasma exposure time is required to break Si-O bonds at SiO2 sites and to activate pore lining CHn groups by removal of H atoms. Sealing efficiency degrades if the pore radius is too large to link the sites of opposite pore walls by Si-N-N-C, Si-N-N-Si or C-N-N-C chains. In this talk, we discuss results from a computational investigation of the sealing efficiency of a porous carbon doped silica films (SiOCH). The Hybrid Plasma Equipment Model provided the fluxes of ions, neutrals and photons onto the surface from He and NH3/Ar ICPs. The sealing mechanism was implemented in the Monte Carlo Feature Profile Model with which profiles of the low-k pores are predicted. Factors affecting the sealing efficiency, such as treatment time, bias, average pore radius and pore radius standard deviation will be discussed.

  15. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

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

  16. 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.

  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. Highly Luminescent Microporous Organic Polymer with Lewis Acidic Boron Sites on the Pore Surface: Ratiometric Sensing and Capture of F(-) Ions.

    PubMed

    Suresh, Venkata M; Bandyopadhyay, Arkamita; Roy, Syamantak; Pati, Swapan K; Maji, Tapas Kumar

    2015-07-20

    Reversible and selective capture/detection of F(-) ions in water is of the utmost importance, as excess intake leads to adverse effects on human health. Highly robust Lewis acidic luminescent porous organic materials have potential for efficient sequestration and detection of F(-) ions. Herein, the rational design and synthesis of a boron-based, Lewis acidic microporous organic polymer (BMOP) derived from tris(4-bromo-2,3,5,6-tetramethylphenyl)boron nodes and diethynylbiphenyl linkers with a pore size of 1.08 nm for selective turn-on sensing and capture of F(-) ion are reported. The presence of a vacant pπ orbital on the boron center of BMOP results in intramolecular charge transfer (ICT) from the linker to boron. BMOP shows selective turn-on blue emission for F(-) ions in aqueous mixtures with a detection limit of 2.6 μM. Strong B-F interactions facilitate rapid sequestration of F(-) by BMOP. The ICT emission of BMOP can be reversibly regenerated by addition of an excess of water, and the polymer can be reused several times. PMID:26074403

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

    PubMed

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

    2016-09-01

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

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

    PubMed Central

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

    2010-01-01

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

  2. Surface modification of nanoporous alumina layers by deposition of Ag nanoparticles. Effect of alumina pore diameter on the morphology of silver deposit and its influence on SERS activity

    NASA Astrophysics Data System (ADS)

    Pisarek, Marcin; Nowakowski, Robert; Kudelski, Andrzej; Holdynski, Marcin; Roguska, Agata; Janik-Czachor, Maria; Kurowska-Tabor, Elżbieta; Sulka, Grzegorz D.

    2015-12-01

    Self-organized Al2O3 nanoporous/nanotubular (Al2O3-NP) oxide layers decorated with silver nanoparticles (Ag-NPs) exhibiting specific properties may serve as attractive SERS substrates for investigating the interactions between an adsorbate and adsorbent, or as stable platforms for detecting various organic compounds. This article presents the influence of the size of the alumina nanopores with a deposit of silver nanoparticles obtained by the magnetron sputtering technique on the morphology of silver film. Moreover, the effect of pore diameter on the intensity of SERS spectra in Ag-NPs/Al2O3-NP/Al composites has also been estimated. For such investigations we used pyridine as a probe molecule, since it has a large cross-section for Raman scattering. To characterize the morphology of the composite oxide layer Ag-NPs/Al2O3-NP/Al, before and after deposition of Ag-NPs by PVD methods (Physical Vapor Deposition), we used scanning electron microscopy (SEM) and atomic force microscopy (AFM). The surface analytical technique of surface-enhanced Raman spectroscopy (SERS) was used to investigate the surface activity of the composite. The results obtained show that, for a carefully controlled amount of Ag (0.020 mg/cm2 - deposited on the top of alumina nanopores whose average size varies from ∼86 nm up to ∼320 nm) in the composites investigated, pore size significantly affects SERS enhancement. We obtained distinctly higher intensities of SERS spectra for substrates with an Ag-NPs deposit having a larger diameter of the alumina nanopores. AFM results suggest that both the lateral and perpendicular distribution of Ag-NPs within and on the top of the largest pores is responsible for the highest SERS activity of the resulting Ag-NPs/Al2O3-NP/Al composite layer, since it produces a variety of cavities and slits which function as resonators for the adsorbed molecules. The Ag-NPs/MeOx-NP/Me composite layers obtained ensure a good reproducibility of the SERS measurements. a

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

    PubMed

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

    1990-03-01

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

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

    SciTech Connect

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

    1993-12-01

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

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

    PubMed

    Gordon, S G; Mourad, A M

    1991-12-01

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

  6. Deposition nucleation viewed as homogeneous or immersion freezing in pores and cavities

    NASA Astrophysics Data System (ADS)

    Marcolli, C.

    2013-06-01

    filled with water. Water in pores can freeze in immersion mode at T > 235 K if the pore walls contain an active site. Pore analysis of clay minerals shows that kaolinites exhibit pore structures with pore diameters of 20-50 nm. The mesoporosity of illites and montmorillonites is characterized by pores with T = 2-5 nm. The number and size of pores is distinctly increased in acid treated montmorillonites like K10. Many clay minerals and mineral dusts show a strong increase in ice nucleation efficiency when temperature is decreased below 235 K. Such an increase is difficult to explain when ice nucleation is supposed to occur by a deposition mechanism, but evident when assuming freezing in pores, because for homogeneous ice nucleation only small pore volumes are needed, while heterogeneous ice nucleation requires larger pore structures to contain at least one active site for immersion nucleation. Together, these pieces of evidence strongly suggest that ice nucleation within pores should be the prevailing freezing mechanism of clay minerals for RHw below water saturation. Extending the analysis to other types of ice nuclei shows that freezing in pores and cracks is probably the prevailing ice nucleation mechanism for glassy and volcanic ash aerosols at RHw below water saturation. Freezing of water in carbon nanotubes might be of significance for ice nucleation by soot aerosols. No case could be identified that gives clear evidence of ice nucleation by water vapor deposition on a solid surface. Inspection of ice nuclei with a close lattice match to ice, such as silver iodide or SnomaxTM, show that for high ice nucleation efficiency below water saturation the presence of impurities or cracks on the surface may be essential. Soluble impurities promote the formation of a liquid phase below water saturation in patches on the surface or as a complete surface layer that offers an environment for immersion freezing. If porous aerosol particles come in contact with semivolatile vapors

  7. HTO and OBT activity concentrations in soil at the historical atmospheric HT release site (Chalk River Laboratories).

    PubMed

    Kim, S B; Bredlaw, M; Korolevych, V Y

    2012-01-01

    Tritium is routinely released by the Chalk River Laboratories (CRL) nuclear facilities. Three International HT release experiments have been conducted at the CRL site in the past. The site has not been disturbed since the last historical atmospheric testing in 1994 and presents an opportunity to assess the retention of tritium in soil. This study is devoted to the measurement of HTO and OBT activity concentration profiles in the subsurface 25 cm of soil. In terms of soil HTO, there is no evidence from the past HT release experiments that HTO was retained. The HTO activity concentration in the soil pore water appears similar to concentrations found in background areas in Ontario. In contrast, OBT activity concentrations in soil at the same site were significantly higher than HTO activity concentrations in soil. Elevated OBT appears to reside in the top layer of the soil (0-5 cm). In addition, OBT activity concentrations in the top soil layer did not fluctuate much with season, again, quite in contrast with soil HTO. This result suggests that OBT activity concentrations retained the signature of the historical tritium releases.

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

    SciTech Connect

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

    2012-02-06

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

  9. Large Enhancement in Electrorheological Activity of Mesoporous Cerium-Doped TIO2 from High Surface Area and Robust Pore Walls

    NASA Astrophysics Data System (ADS)

    Yin, Jianbo; Zhao, Xiaopeng

    Considering the importance of large interfacial or surface polarization to strong electrorheological (ER) effect, we developed a high surface area mesoporous doped TiO2 ER material by using block-copolymer. By comparing the ER experiments between samples with mesopore and without mesopore, we demonstrate a very large enhancement in ER activity of mesoporous ER material and its yield stress is 100 times that of the pure TiO2 ER material and 5-8 times that of single doped TiO2 without mesoporous structure. We give a preliminary discussion about the improvement in ER activity based on previous dielectric analysis.

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

    PubMed

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

    1986-05-01

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

  11. Target cell death triggered by cytotoxic T lymphocytes: a target cell mutant distinguishes passive pore formation and active cell suicide mechanisms.

    PubMed Central

    Ucker, D S; Wilson, J D; Hebshi, L D

    1994-01-01

    The role of the target cell in its own death mediated by cytotoxic T lymphocytes (CTL) has been controversial. The ability of the pore-forming granule components of CTL to induce target cell death directly has been taken to suggest an essentially passive role for the target. This view of CTL-mediated killing ascribes to the target the single role of providing an antigenic stimulus to the CTL; this signal results in the vectoral degranulation and secretion of pore-forming elements onto the target. On the other hand, by a number of criteria, target cell death triggered by CTL appears fundamentally different from death resulting from membrane damage and osmotic lysis. CTL-triggered target cell death involves primary internal lesions of the target cell that reflect a physiological cell death process. Orderly nuclear disintegration, including lamin phosphorylation and solubilization, chromatin condensation, and genome digestion, are among the earliest events, preceding the loss of plasma membrane integrity. We have tested directly the involvement of the target cell in its own death by examining whether we could isolate mutants of target cells that have retained the ability to be recognized by and provide an antigenic stimulus to CTL while having lost the capacity to respond by dying. Here, we describe one such mutant, BW87. We have used this CTL-resistant mutant to analyze the mechanisms of CTL-triggered target cell death under a variety of conditions. The identification of a mutable target cell element essential for the cell death response to CTL provides genetic evidence that target cell death reflects an active cell suicide process similar to other physiological cell deaths. PMID:8264610

  12. Pore morphologies of root induced biopores from single pore to network scale investigated by XRCT

    NASA Astrophysics Data System (ADS)

    Peth, Stephan; Wittig, Marlen C.; Uteau Puschmann, Daniel; Pagenkemper, Sebastian; Haas, Christoph; Holthusen, Dörthe; Horn, Rainer

    2015-04-01

    Biopores are assumed to be an important factor for nutrient acquisition by providing biologically highly active soil-root interfaces to re-colonizing roots and controlling oxygen and water flows at the pedon scale and within the rhizosphere through the formation of branching channel networks which potentially enhance microbial turnover processes. Characteristic differences in pore morphologies are to be expected depending on the genesis of biopores which, for example, can be earthworm-induced or root-induced or subsequently modified by one of the two. Our understanding of biophysical interactions between plants and soil can be significantly improved by quantifying 3D biopore architectures across scales ranging from single biopores to pedon scale pore networks and linking pore morphologies to microscale measurements of transport processes (e.g. oxygen diffusion). While a few studies in the past have investigated biopore networks on a larger scale yet little is known on the micro-morphology of root-induces biopores and their associated rhizosphere. Also little data is available on lateral transport of oxygen through the rhizosphere which will strongly influence microbial turnover processes and consequently control the release and uptake of nutrients. This paper highlights results gathered within a research unit on nutrient acquisition from the subsoil. Here we focus on X-ray microtomography (XRCT) studies ranging from large soil columns (70 cm length and 20 cm diameter) to individual biopores and its surrounding rhizosphere. Samples were collected from sites with different preceding crops (fescue, chicory, alfalfa) and various cropping durations (1-3 years). We will present an approach for quantitative image analysis combined with micro-sensor measurements of oxygen diffusion and spatial gradients of O2 partial pressures to relate pore structure with transport functions. Implications of various biopore architectures for the accessibility of nutrient resources in

  13. Short, Synthetic Cationic Peptides Have Antibacterial Activity against Mycobacterium smegmatis by Forming Pores in Membrane and Synergizing with Antibiotics

    PubMed Central

    Gupta, Kajal; Singh, Sameer; van Hoek, Monique L.

    2015-01-01

    Multicellular organisms are constantly exposed to a multitude of pathogenic microbes. Infection is inhibited in vivo by the innate and adaptive immune system. Mycobacterium species have emerged that are resistant to most antibiotics. We identified several naturally occurring cationic antimicrobial peptides that were active at low micromolar concentrations against Mycobacterium smegmatis. Human-derived cathelicidin LL-37 is well characterized and studied against M. smegmatis; we compared LL-37 with Chinese cobra-derived cathelicidin NA-CATH and mouse cathelicidin (mCRAMP). Two synthetic 11-residue peptides (ATRA-1A and ATRA-2) containing variations of a repeated motif within NA-CATH were tested for their activity against M. smegmatis along with a short synthetic peptide derivative from the human beta-defensin hBD3 (hBD3-Pep4). We hypothesized that these smaller synthetic peptides may demonstrate antimicrobial effectiveness with shorter length (and at less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds or use in combination with antibiotics. These peptides have antimicrobial activity with EC50 ranging from 0.05 to 1.88 μg/mL against Mycobacterium smegmatis. The ATRA-1A short peptide was found to be the most effective antimicrobial peptide (AMP) (EC50 = 0.05 μg/mL). High bactericidal activity correlated with bacterial membrane depolarization and permeabilization activities. The efficacy of the peptides was further analyzed through Minimal Inhibitory Concentration (MIC) assays. The MICs were determined by the microdilution method. The peptide mCRAMP showed the best MIC activity at 15.6 μg/mL. Neither of the effective short synthetic peptides demonstrated synergy with the antibiotic rifampicin, although both demonstrated synergy with the cyclic peptide antibiotic polymyxin B. The peptides LL-37 and mCRAMP displayed synergism with rifampicin in MIC assays, whereas antibiotic polymyxin B displayed synergism

  14. 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.

  15. A unique alkaline pH-regulated and fatty acid-activated tandem pore domain potassium channel (K2P) from a marine sponge

    PubMed Central

    Wells, Gregory D.; Tang, Qiong-Yao; Heler, Robert; Tompkins-MacDonald, Gabrielle J.; Pritchard, Erica N.; Leys, Sally P.; Logothetis, Diomedes E.; Boland, Linda M.

    2012-01-01

    SUMMARY A cDNA encoding a potassium channel of the two-pore domain family (K2P, KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK2P cannot be placed into any of the established functional groups of mammalian K2P channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK2P. In whole cells, non-inactivating, voltage-independent, outwardly rectifying K+ currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC50 ∼30 μmol l–1), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK2P but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K2P channels, the sponge K2P channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pKa 8.18) activated the AquK2P channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K2P channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K2P channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA. PMID:22723483

  16. Active sites in char gasification: Final technical report

    SciTech Connect

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

    1987-09-01

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

  17. Effects of amino acid mutations in the pore-forming domain of the hemolytic lectin CEL-III.

    PubMed

    Nagao, Tomonao; Masaki, Risa; Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

    2016-10-01

    The hemolytic lectin CEL-III forms transmembrane pores in the membranes of target cells. A study on the effect of site-directed mutation at Lys405 in domain 3 of CEL-III indicated that replacements of this residue by relatively smaller residues lead to a marked increase in hemolytic activity, suggesting that moderately destabilizing domain 3 facilitates formation of transmembrane pores through conformational changes.

  18. Effects of amino acid mutations in the pore-forming domain of the hemolytic lectin CEL-III.

    PubMed

    Nagao, Tomonao; Masaki, Risa; Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

    2016-10-01

    The hemolytic lectin CEL-III forms transmembrane pores in the membranes of target cells. A study on the effect of site-directed mutation at Lys405 in domain 3 of CEL-III indicated that replacements of this residue by relatively smaller residues lead to a marked increase in hemolytic activity, suggesting that moderately destabilizing domain 3 facilitates formation of transmembrane pores through conformational changes. PMID:27101707

  19. 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.

  20. Active Sites Environmental Monitoring Program: FY 1991 report

    SciTech Connect

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

    1992-11-01

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

  1. Inhibition and active-site modelling of prolidase.

    PubMed

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

    1989-03-15

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

  2. Salt marsh pore water geochemistry does not correlate with microbial community structure

    NASA Astrophysics Data System (ADS)

    Koretsky, Carla M.; Van Cappellen, Philippe; DiChristina, Thomas J.; Kostka, Joel E.; Lowe, Kristi L.; Moore, Charles M.; Roychoudhury, Alakendra N.; Viollier, Eric

    2005-01-01

    Spatial and temporal trends in pore water geochemistry and sediment microbial community structure are compared at three intertidal sites of a saltmarsh on Sapelo Island, GA. The sites include a heavily bioturbated, unvegetated creek bank, a levee with dense growth of Spartina alterniflora, and a more sparsely vegetated ponded marsh site. The redox chemistry of the pore waters ranges from sulfide-dominated at the ponded marsh site to suboxic at the creek bank site. At the three sites, the vertical redox stratification of the pore waters is more compressed in summer than in winter. The trends in redox chemistry reflect opposing effects of sediment respiration and pore water irrigation. Intense and deep burrowing activity by fiddler crabs at the creek bank site results in the efficient oxidation of reduced byproducts of microbial metabolism and, hence, the persistence of suboxic conditions to depths of 50 cm below the sediment surface. Increased supply of labile organic substrates at the vegetated sites promotes microbial degradation processes, leading to sharper redox gradients. At the levee site, this is partly offset by the higher density and deeper penetration of roots and macrofaunal burrows. Surprisingly, the microbial community structure shows little correlation with the variable vertical redox zonation of the pore waters across the saltmarsh. At the three sites, the highest population densities of aerobic microorganisms, iron- plus manganese-reducing bacteria, and sulfate reducers coexist within the upper 10 cm of sediment. The absence of a clear vertical separation of these microorganisms is ascribed to the high supply of labile organic matter and intense mixing of the topmost sediment via bioturbation.

  3. Velocities in Solar Pores

    NASA Astrophysics Data System (ADS)

    Balasubramaniam, K. S.; Keil, S. L.; Smaldone, L. A.

    1996-05-01

    We investigate the three dimensional structure of solar pores and their surroundings using high spatial and spectral resolution data. We present evidence that surface velocities decrease around pores with a corresponding increase in the line-of-sight (LOS) velocities. LOS velocities in pores increase with the strength of the magnetic field. Surface velocities show convergence toward a weak downflow which appear to trace boundaries resembling meso-granular and super granular flows. The observed magnetic fields in the pores appear near these boundaries. We analyze the vertical velocity structure in pores and show that they generally have downflows decreasing exponentially with height, with a scale height of about 90 km. Evidence is also presented for the expanding nature of flux tubes. Finally we describe a phenomenological model for pores. This work was supported by AFOSR Task 2311G3. LAS was partially supported by the Progetto Nazionale Astrofisica e Fisica Cosmica of MURST and Scambi Internazionali of the Universita degli Studi di Napoli Frederico II. National Solar Observatory, NOAO, is operated for the National Science Foundation by AURA, Inc.

  4. A Non-inactivating High-voltage-activated Two-Pore Na+ Channel that Supports Ultra-long Action Potentials and Membrane Bistability

    PubMed Central

    Cang, Chunlei; Aranda, Kimberly; Ren, Dejian

    2014-01-01

    Action potentials (APs) are fundamental cellular electrical signals. The genesis of short APs lasting milliseconds is well understood. Ultra-long APs (ulAPs) lasting seconds to minutes also occur in eukaryotic organisms, but their biological functions and mechanisms of generation are largely unknown. Here, we identify TPC3, a previously uncharacterized member of the two-pore channel protein family, as a new voltage-gated Na+ channel (NaV) that generates ulAPs, and that establishes membrane potential bistability. Unlike the rapidly inactivating NaVs that generate short APs in neurons, TPC3 has a high activation threshold, activates slowly, and does not inactivate—three properties that help generate long-lasting APs and guard the membrane against unintended perturbation. In amphibian oocytes, TPC3 forms a channel similar to channels induced by depolarization and sperm entry into eggs. TPC3 homologs are present in plants and animals, and they may be important for cellular processes and behaviors associated with prolonged membrane depolarization. PMID:25256615

  5. Investigation of the pore structure and morphology of cellulose acetate membranes using small-angle neutron scattering. 1: Cellulose acetate active layer membranes

    SciTech Connect

    Kulkarni, S.; Krause, S. ); Wignall, G.D. . Solid State Div.); Hammouda, B. . Center for High Resolution Neutron Scattering)

    1994-11-07

    The structure of ultrathin cellulose acetate membranes, known as active layer membranes, has been investigated using small-angle neutron scattering. These membranes are known to have structural and functional similarity to the surface or skin layer in commercial reverse-osmosis (RO) membranes and hence are useful model systems for understanding the structure of the RO membrane skin layer. Active layer membranes were studied after swelling them with either D[sub 2]O or CD[sub 3]OD. The results in both cases clearly indicated the presence of very small (10--20 [angstrom]) porous structures in the membrane. The presence of such pores has been a subject of long-standing controversy in this area. The data were analyzed using a modified Debye-Bueche analysis and the resultant membrane structure was seen to agree well with structural information from electron microscopic studies. Finally, a possible explanation for the differences in scattering observed between the D[sub 2]O swollen membranes and the CD[sub 3]OD swollen membranes has been presented.

  6. A non-inactivating high-voltage-activated two-pore Na+ channel that supports ultra-long action potentials and membrane bistability

    NASA Astrophysics Data System (ADS)

    Cang, Chunlei; Aranda, Kimberly; Ren, Dejian

    2014-09-01

    Action potentials (APs) are fundamental cellular electrical signals. The genesis of short APs lasting milliseconds is well understood. Ultra-long APs (ulAPs) lasting seconds to minutes also occur in eukaryotic organisms, but their biological functions and mechanisms of generation are largely unknown. Here, we identify TPC3, a previously uncharacterized member of the two-pore channel protein family, as a new voltage-gated Na+ channel (NaV) that generates ulAPs, and that establishes membrane potential bistability. Unlike the rapidly inactivating NaVs that generate short APs in neurons, TPC3 has a high activation threshold, activates slowly and does not inactivate—three properties that help generate long-lasting APs and guard the membrane against unintended perturbation. In amphibian oocytes, TPC3 forms a channel similar to channels induced by depolarization and sperm entry into eggs. TPC3 homologues are present in plants and animals, and they may be important for cellular processes and behaviours associated with prolonged membrane depolarization.

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

    PubMed Central

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

    2016-01-01

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

  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. Radiocarbon Evidence for Active Turnover of Pore-Water Dissolved Organic Carbon in the Methanogenic and Sulfate-Methane-Transition Zones of Santa Barbara Basin Sediments

    NASA Astrophysics Data System (ADS)

    Komada, T.; Li, H. L.; Cada, A. K.; Burdige, D.; Magen, C.; Chanton, J.; Grose, A. M.

    2014-12-01

    Diverse metabolic activities have been documented in the deep biosphere. However, how these activities affect carbon cycling in the subsurface, and how they in turn affect the marine and global cycles of carbon are still unclear. Here we present natural-abundance 14C and 13C data from the uppermost 4.5 m of the sediments of the Santa Barbara Basin, California Borderland, showing active turnover of dissolved organic carbon (DOC) within, and immediately below, the sulfate-methane transition zone (SMTZ; ~1.25 m). DOC concentrations increased with depth throughout the core, indicating net production within the sediment column. Enhanced DOC production was observed near the sediment-water interface, and also at ~30 cm below the SMTZ (~1.55 m). ∆14C values of DOC increased across the sediment-water interface, then decreased with depth, consistent with net production of modern DOC near the sediment-water interface, and input of 14C-depleted DOC from deeper horizons. An isotope mixing plot constructed with these data shows that the DOC diffusing upward at the base of the core is devoid of 14C, yet the DOC diffusing into and out of the SMTZ is relatively enriched (-460‰ and -300‰, respectively). This difference in 14C content of the DOC flux can only be reconciled if the following two are occurring within, and immediately below, the SMTZ: (1) >90% of the 14C-dead basal DOC flux is removed from the pore water (by, e.g., oxidation, fermentation, methanogenesis, precipitation), and (2) this DOC is replaced by material produced in this region at a rate that exceeds the upward basal flux. The 14C and 13C signatures suggest sedimentary organic matter to be the dominant source of DOC in process (2). Our data provide a unique insight into the active transformation of DOC and sedimentary organic matter in the subsurface.

  10. Electrostatic fields in the active sites of lysozymes.

    PubMed

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

    1989-07-01

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

  11. Histidine at the active site of Neurospora tyrosinase.

    PubMed

    Pfiffner, E; Lerch, K

    1981-10-13

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

  12. The influence of small mammal burrowing activity on water storage at the Hanford Site

    SciTech Connect

    Landeen, D.S.

    1994-09-01

    The amount and rate at which water may penetrate a protective barrier and come into contact with buried radioactive waste is a major concern. Because burrowing animals eventually will reside on the surface of any protective barrier, the effect these burrow systems may have on the loss or retention of water needs to be determined. The first section of this document summarizes the known literature relative to small mammals and the effects that burrowing activities have on water distribution, infiltration, and the overall impact of burrows on the ecosystem. Topics that are summarized include burrow air pressures, airflow, burrow humidity, microtopography, mounding, infiltration, climate, soil evaporation, and discussions of large pores relative to water distribution. The second section of this document provides the results of the study that was conducted at the Hanford Site to determine what effect small mammal burrows have on water storage. This Biointrusion task is identified in the Permanent Isolation Surface Barrier Development Plan in support of protective barriers. This particular animal intrusion task is one part of the overall animal intrusion task identified in Animal Intrusion Test Plan.

  13. Geosmin and 2-methylisoborneol removal using superfine powdered activated carbon: shell adsorption and branched-pore kinetic model analysis and optimal particle size.

    PubMed

    Matsui, Yoshihiko; Nakao, Soichi; Taniguchi, Takuma; Matsushita, Taku

    2013-05-15

    2-Methylisoborneol (MIB) and geosmin are naturally occurring compounds responsible for musty-earthy taste and odor in public drinking-water supplies, a severe problem faced by many utilities throughout the world. In this study, we investigated adsorptive removal of these compounds by superfine powdered activation carbon (SPAC, particle size <1 μm) produced by novel micro-grinding of powdered activated carbon; we also discuss the optimization of carbon particle size to efficiently enhance the adsorptive removal. After grinding, the absorptive capacity remained unchanged for a 2007 carbon sample and was increased for a 2010 carbon sample; the capacity increase was quantitatively described by the shell adsorption model, in which MIB and geosmin adsorbed more in the exterior of a carbon particle than in the center. The extremely high uptake rates of MIB and geosmin by SPAC were simulated well by a combination of the branched-pore kinetic model and the shell adsorption model, in which intraparticle diffusion through macropores was followed by diffusion from macropore to micropore. Simulations suggested that D40 was on the whole the best characteristic diameter to represent a size-disperse group of adsorbent particles; D40 is the diameter through which 40% of the particles by volume pass. Therefore, D40 can be used as an index for evaluating the improvement of adsorptive removal that resulted from pulverization. The dose required for a certain percentage removal of MIB or geosmin decreased linearly with carbon particle size (D40), but the dose reduction became less effective as the activated carbon was ground down to smaller sizes around a critical value of D40. For a 60-min contact time, critical D40 was 2-2.5 μm for MIB and 0.4-0.5 μm for geosmin. The smaller critical D40 was when the shorter the carbon-water contact time was or the slower the intraparticle mass transfer rate of an adsorbate was. PMID:23528781

  14. Geosmin and 2-methylisoborneol removal using superfine powdered activated carbon: shell adsorption and branched-pore kinetic model analysis and optimal particle size.

    PubMed

    Matsui, Yoshihiko; Nakao, Soichi; Taniguchi, Takuma; Matsushita, Taku

    2013-05-15

    2-Methylisoborneol (MIB) and geosmin are naturally occurring compounds responsible for musty-earthy taste and odor in public drinking-water supplies, a severe problem faced by many utilities throughout the world. In this study, we investigated adsorptive removal of these compounds by superfine powdered activation carbon (SPAC, particle size <1 μm) produced by novel micro-grinding of powdered activated carbon; we also discuss the optimization of carbon particle size to efficiently enhance the adsorptive removal. After grinding, the absorptive capacity remained unchanged for a 2007 carbon sample and was increased for a 2010 carbon sample; the capacity increase was quantitatively described by the shell adsorption model, in which MIB and geosmin adsorbed more in the exterior of a carbon particle than in the center. The extremely high uptake rates of MIB and geosmin by SPAC were simulated well by a combination of the branched-pore kinetic model and the shell adsorption model, in which intraparticle diffusion through macropores was followed by diffusion from macropore to micropore. Simulations suggested that D40 was on the whole the best characteristic diameter to represent a size-disperse group of adsorbent particles; D40 is the diameter through which 40% of the particles by volume pass. Therefore, D40 can be used as an index for evaluating the improvement of adsorptive removal that resulted from pulverization. The dose required for a certain percentage removal of MIB or geosmin decreased linearly with carbon particle size (D40), but the dose reduction became less effective as the activated carbon was ground down to smaller sizes around a critical value of D40. For a 60-min contact time, critical D40 was 2-2.5 μm for MIB and 0.4-0.5 μm for geosmin. The smaller critical D40 was when the shorter the carbon-water contact time was or the slower the intraparticle mass transfer rate of an adsorbate was.

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

    PubMed

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

    1995-02-28

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

  16. The copper active site of CBM33 polysaccharide oxygenases.

    PubMed

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

    2013-04-24

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

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

    PubMed Central

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

    1996-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2004-04-01

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

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

    PubMed

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

    2015-07-21

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    SciTech Connect

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

    1991-09-10

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

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

    PubMed

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

    2016-04-22

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

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

    SciTech Connect

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

    1986-03-01

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

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

    PubMed

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

    2016-04-22

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

  7. Toxicity of sediments and pore water from Brunswick Estuary, Georgia

    USGS Publications Warehouse

    Winger, P.V.; Lasier, P.J.; Geitner, H.

    1993-01-01

    A chlor-alkali plant in Brunswick, Georgia, USA, discharged >2 kg mercury/d into a tributary of the Turtle River-Brunswick Estuary from 1966 to 1971. Mercury concentrations in sediments collected in 1989 along the tributary near the chlor-alkali plant ranged from 1 to 27 ug/g (dry weight), with the highest concentrations found in surface (0?8 cm) sediments of subtidal zones in the vicinity of the discharge site. Toxicity screening in 1990 using Microtox? bioassays on pore water extracted on site from sediments collected at six stations distributed along the tributary indicated that pore water was highly toxic near the plant discharge. Ten-day toxicity tests on pore water from subsequent sediment samples collected near the plant discharge confirmed high toxicity to Hyalella azteca, and feeding activity was significantly reduced in whole-sediment tests. In addition to mercury in the sediments, other metals (chromium, lead, and zinc) exceeded 50 ug/g, and polychlorobiphenyl (PCB) concentrations ranged from 67 to 95 ug/g. On a molar basis, acid-volatile sulfide concentrations (20?45 umol/g) in the sediments exceeded the metal concentrations. Because acid-volatile sulfides bind with cationic metals and form metal sulfides, which are generally not bioavailable, toxicities shown by these sediments were attributed to the high concentrations of PCBs and possibly methylmercury.

  8. Activation of the Human Epithelial Sodium Channel (ENaC) by Bile Acids Involves the Degenerin Site.

    PubMed

    Ilyaskin, Alexandr V; Diakov, Alexei; Korbmacher, Christoph; Haerteis, Silke

    2016-09-16

    The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin ion channel family, which also includes the bile acid-sensitive ion channel (BASIC). So far little is known about the effects of bile acids on ENaC function. ENaC is probably a heterotrimer consisting of three well characterized subunits (αβγ). In humans, but not in mice and rats, an additional δ-subunit exists. The aim of this study was to investigate the effects of chenodeoxycholic, cholic, and deoxycholic acid in unconjugated (CDCA, CA, and DCA) and tauro-conjugated (t-CDCA, t-CA, t-DCA) form on human ENaC in its αβγ- and δβγ-configuration. We demonstrated that tauro-conjugated bile acids significantly stimulate ENaC in the αβγ- and in the δβγ-configuration. In contrast, non-conjugated bile acids have a robust stimulatory effect only on δβγENaC. Bile acids stimulate ENaC-mediated currents by increasing the open probability of active channels without recruiting additional near-silent channels known to be activated by proteases. Stimulation of ENaC activity by bile acids is accompanied by a significant reduction of the single-channel current amplitude, indicating an interaction of bile acids with a region close to the channel pore. Analysis of the known ASIC1 (acid-sensing ion channel) crystal structure suggested that bile acids may bind to the pore region at the degenerin site of ENaC. Substitution of a single amino acid residue within the degenerin region of βENaC (N521C or N521A) significantly reduced the stimulatory effect of bile acids on ENaC, suggesting that this site is critical for the functional interaction of bile acids with the channel. PMID:27489102

  9. 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.

  10. 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.

  11. Effect of the ostreolysin A/pleurotolysin B pore-forming complex on neuroblastoma cell morphology and intracellular Ca²⁺ activity.

    PubMed

    Vrecl, Milka; Babnik, Monika; Diacci, Uroš; Benoit, Evelyne; Frangež, Robert

    2015-04-01

    Ostreolysin A (OlyA) and pleurotolysin B (PlyB), isolated from edible oyster mushrooms, form a cytolytic complex (OlyA/PlyB) in membrane cells that causes respiratory arrest. This study evaluated the mechanisms underlying cytotoxic OlyA/PlyB activity in neuroblastoma NG108-15 cells. Confocal microscopy with morphometric analysis revealed that OlyA/PlyB increased the 3-dimensional projected area of differentiated cells. Iso-osmotic replacement of NaCl by sucrose or Na-isethionate prevented the cellular swelling. This suggests that formation of cellular edema requires the presence of Na(+) and/or Cl(-) in the extracellular space and may be related to an influx of Na(+) and/or a shift in Cl(-), which induce a marked influx of water that is ultimately responsible for cellular swelling. In addition, extracellular Ca(2+) moderately contributed to the swelling because benzamil (10 µM), a 3Na(+)/Ca(2+) exchange (NCX) inhibitor, and Ca(2+)-free medium partially prevented this response. Fluorometric measurements revealed that OlyA/PlyB, at approximately 15-fold higher concentrations, increased the intracellular Ca(2+) activity [Ca(2+)]i. This increase was dependent on the presence of Na(+) and Ca(2+) in the external medium and was sensitive to benzamil. It is thus likely that a switch in the NCX mode, associated with the de novo formation of non-selective ion pores by OlyA/PlyB in cellular plasma membranes, plays an important role in this effect. Overall, OlyA/PlyB affects neuroblastoma cell morphology and Ca(2+) homeostasis to influence the toxin-induced respiratory arrest. PMID:25556216

  12. Fusion pore regulation of transmitter release.

    PubMed

    Fernández-Peruchena, Carlos; Navas, Sergio; Montes, María A; Alvarez de Toledo, Guillermo

    2005-09-01

    During the last decade a wealth of new information about the properties of the exocytotic fusion pore is changing our current view of exocytosis. The exocytotic fusion pore, a necessary stage before the full merging of the vesicle membrane with the plasma membrane, is becoming a key cellular structure that might critically control the amount of neurotransmitter released into the synaptic cleft and that can be subjected to control by second messengers and phosphorylated proteins. Fusion pores form, expand to fully merge membranes, or can close leaving an intact and identical synaptic vesicle in place for a new round of exocytosis. Transient formation of fusion pores is the mechanistic representation of the "kiss-and-run" hypothesis of transmitter release and offers new alternatives for synaptic vesicle recycling besides to the classical mechanism mediated by clathrin coat endocytosis. For vesicle recycling transient fusion pores ensures a fast mechanism for maintaining an active pool of synaptic vesicles. The size reached by transient fusion pores and the time spent on the open state can determine the release of subquantal synaptic transmission, which could be a mechanism of synaptic potentiation. In this review we will described the electrophysiological and fluorescence methods that contribute to further explore the biophysical properties of the exocytotic fusion pore and the relevant experiments obtained by these methods.

  13. 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.

  14. Formation and decay of rudimentary penumbra around a pore

    SciTech Connect

    Watanabe, Hiroko; Kitai, Reizaburo; Otsuji, Kenichi

    2014-12-01

    We analyze the evolution of a pore in the active region NOAA 10940 using the data obtained by the Hinode satellite on 2007 February 3. The pore we analyzed showed the formation of a rudimentary penumbra structure, succeeded by an abrupt disappearance after about 5 hr. The pore had an approximate radius of 3.5 Mm and a total magnetic flux of 3.0 × 10{sup 19} Mx, which is a little smaller than the necessary magnetic flux for penumbral formation supposed by Rucklidge et al. (1-1.5 × 10{sup 20} Mx). Our observation describes a rare phenomenon which was in the unstable phase between a pore and a sunspot. The area of the dark umbra gradually decreased when the rudimentary penumbral filaments formed the penumbral structure, meaning that the penumbra develops at the expense of the umbral magnetic flux. This statement was confirmed by a rough estimation of the magnetic flux variation observed by the Hinode Fe I magnetogram. Five hours after the formation phase, the decay phase began. In this decaying phase, multiple opposite polarity patches are found to appear in the exterior of the pore (a different location from the penumbra formation site). We interpret these opposite polarities as signatures of the horizontal magnetic field, which preferably appears in the course of the unstable reconfiguration of the magnetic field structure. During the course of the disappearance of the penumbra, the horizontal penumbral field seems to become vertical because of the dark umbral area that recovered by about 10%.

  15. 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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  20. Mutagenesis and functional analysis of the pore-forming toxin HALT-1 from Hydra magnipapillata.

    PubMed

    Liew, Yvonne Jing Mei; Soh, Wai Tuck; Jiemy, William Febry; Hwang, Jung Shan

    2015-02-01

    Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1-4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.

  1. Comparison of unimodal versus bimodal pore catalysts in residues hydrotreating

    SciTech Connect

    Absi-Halabi, M.; Stanislaus, A.; Al-Zaid, H.

    1994-12-31

    Catalyst pore structure is a critical factor influencing the performance of residues hydroprocessing catalysts. The effect is reflected in both hydrodesulfurization activity of the catalyst and its rate of deactivation. In this paper, the pore size distributions of two categories of catalysts, unimodal and bimodal, were systematically varied. Performance evaluation tests in a fixed bed reactor using vacuum residues under conditions comparable to typical refinery operations were conducted. Two series of unimodal and bimodal catalyst extrudates were prepared starting from boehmite gel, whereby the pore structure was systematically varied using hydrothermal treatment and organic additives. For the unimodal catalysts, the pore maxima ranged between 50 and 500 {angstrom} with 70--80% of the pore volume in the desired pore diameter range. The bimodal catalysts had narrow pores with pore diameters less than 100 {angstrom} and wide pres with pore diameter around 5,000 {angstrom}. For bimodal catalyst, an increase in the average wide pore diameter, while maintaining the narrow pore constant, had no significant effect on the catalyst performance. For monomodal catalyst, the activity of the catalyst was noted to have an optimum between 150--350 {angstrom} diameter. Furthermore, the performance of the catalyst concerning its desulfurization activity and deactivation was superior to that of the bimodal catalysts.

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

    PubMed

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

    2015-08-01

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

  3. Pore formation by Cry toxins.

    PubMed

    Soberón, Mario; Pardo, Liliana; Muñóz-Garay, Carlos; Sánchez, Jorge; Gómez, Isabel; Porta, Helena; Bravo, Alejandra

    2010-01-01

    Bacillus thuringiensis (Bt) bacteria produce insecticidal Cry and Cyt proteins used in the biological control of different insect pests. In this review, we will focus on the 3d-Cry toxins that represent the biggest group of Cry proteins and also on Cyt toxins. The 3d-Cry toxins are pore-forming toxins that induce cell death by forming ionic pores into the membrane of the midgut epithelial cells in their target insect. The initial steps in the mode of action include ingestion of the protoxin, activation by midgut proteases to produce the toxin fragment and the interaction with the primary cadherin receptor. The interaction of the monomeric CrylA toxin with the cadherin receptor promotes an extra proteolytic cleavage, where helix alpha-1 of domain I is eliminated and the toxin oligomerization is induced, forming a structure of 250 kDa. The oligomeric structure binds to a secondary receptor, aminopeptidase N or alkaline phosphatase. The secondary receptor drives the toxin into detergent resistant membrane microdomains formingpores that cause osmotic shock, burst of the midgut cells and insect death. Regarding to Cyt toxins, these proteins have a synergistic effect on the toxicity of some Cry toxins. Cyt proteins are also proteolytic activated in the midgut lumen of their target, they bind to some phospholipids present in the mosquito midgut cells. The proposed mechanism of synergism between Cry and Cyt toxins is that Cyt1Aa function as a receptor for Cry toxins. The Cyt1A inserts into midgut epithelium membrane and exposes protein regions that are recognized by Cry11Aa. It was demonstrated that this interaction facilitates the oligomerization of Cry11Aa and also its pore formation activity.

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

    PubMed

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

    2012-09-01

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

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

    PubMed Central

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

    2014-01-01

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

  6. Sweat pore reactivity as a surrogate measure of sympathetic nervous system activity in trauma-exposed individuals with and without posttraumatic stress disorder.

    PubMed

    Familoni, Babajide O; Gregor, Kristin L; Dodson, Thomas S; Krzywicki, Alan T; Lowery, Bobby N; Orr, Scott P; Suvak, Michael K; Rasmusson, Ann M

    2016-09-01

    Stress analysis by FLIR (forward-looking infrared) evaluation (SAFE) has been demonstrated to monitor sweat pore activation (SPA) as a novel surrogate measure of sympathetic nervous system (SNS) activity in a normal population. SNS responses to a series of 15 1-s, 82 dB, white noise bursts were measured by skin conductance (SC) and SAFE monitoring of SPA on the fingers (FiP) and face (FaP) in 10 participants with posttraumatic stress disorder (PTSD) and 16 trauma-exposed participants without PTSD (Mage  = 48.92 ± 12.00 years; 26.9% female). Within participants, SC and FiP responses across trials were strongly correlated (r = .92, p < .001). Correlations between SC and FaP (r = .76, p = .001) and between FiP and FaP (r = .47, p = .005) were smaller. The habituation of SNS responses across the 15 trials was substantial (SC: d = -2.97; FiP: d = -2.34; FaP: d = -1.02). There was a strong correlation between habituation effects for SC and FiP (r = .76, p < .001), but not for SC and FaP (r = .15, p = .45) or FiP and FaP (r = .29, p = .16). Participants with PTSD showed larger SNS responses to the first loud noise than those without PTSD. PTSD reexperiencing symptoms assessed by the PTSD Checklist on the day of testing were associated with the SNS responses to the first loud noise measured by SC (d = 1.19) and FiP (d = .99), but not FaP (d = .10). This study confirms convergence of SAFE and SC as valid measures of SNS activity. SAFE FiP and SC responses were highly predictive of self-rated PTSD reexperiencing symptoms. SAFE may offer an attractive alternative for applications in PTSD and similar populations. PMID:27286885

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

    PubMed

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

    2015-05-01

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

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

    PubMed

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

    2015-02-24

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

  9. Low pore connectivity in natural rock.

    PubMed

    Hu, Qinhong; Ewing, Robert P; Dultz, Stefan

    2012-05-15

    As repositories for CO(2) and radioactive waste, as oil and gas reservoirs, and as contaminated sites needing remediation, rock formations play a central role in energy and environmental management. The connectivity of the rock's porespace strongly affects fluid flow and solute transport. This work examines pore connectivity and its implications for fluid flow and chemical transport. Three experimental approaches (imbibition, tracer concentration profiles, and imaging) were used in combination with network modeling. In the imbibition results, three types of imbibition slope [log (cumulative imbibition) vs. log (imbibition time)] were found: the classical 0.5, plus 0.26, and 0.26 transitioning to 0.5. The imbibition slope of 0.26 seen in Indiana sandstone, metagraywacke, and Barnett shale indicates low pore connectivity, in contrast to the slope of 0.5 seen in the well-connected Berea sandstone. In the tracer profile work, rocks exhibited different distances to the plateau porosity, consistent with the pore connectivity from the imbibition tests. Injection of a molten metal into connected pore spaces, followed by 2-D imaging of the solidified alloy in polished thin sections, allowed direct assessment of pore structure and lateral connection in the rock samples. Pore-scale network modeling gave results consistent with measurements, confirming pore connectivity as the underlying cause of both anomalous behaviors: imbibition slope not having the classical value of 0.5, and accessible porosity being a function of distance from the edge. A poorly connected porespace will exhibit anomalous behavior in fluid flow and chemical transport, such as a lower imbibition slope (in air-water system) and diffusion rate than expected from classical behavior.

  10. Visualizing and Quantifying Bioaccessible Pores in Field-Aged Petroleum Hydrocarbon-Contaminated Clay Soils Using Synchrotron-based X-ray Computed Tomography

    NASA Astrophysics Data System (ADS)

    Chang, W.; Kim, J.; Zhu, N.; McBeth, J. M.

    2015-12-01

    Microbial hydrocarbon degradation is environmentally significant and applicable to contaminated site remediation practices only when hydrocarbons (substrates) are physically bioaccessible to bacteria in soil matrices. Powerful X-rays are produced by synchrotron radiation, allowing for bioaccessible pores in soil (larger than 4 microns), where bacteria can be accommodated, colonize and remain active, can be visualized at a much higher resolution. This study visualized and quantified such bioaccessible pores in intact field-aged, oil-contaminated unsaturated soil fractions, and examined the relationship between the abundance of bioaccessible pores and hydrocarbon biodegradation. Using synchrotron-based X-ray Computed Tomography (CT) at the Canadian Light Source, a large dataset of soil particle characteristics, such as pore volumes, surface areas, number of pores and pore size distribution, was generated. Duplicate samples of five different soil fractions with different soil aggregate sizes and water contents (13, 18 and 25%) were examined. The method for calculating the number and distribution of bioaccessible pores using CT images was validated using the known porosity of Ottawa sand. This study indicated that the distribution of bioaccessible pore sizes in soil fractions are very closely related to microbial enhancement. A follow-up aerobic biodegradation experiment for the soils at 17 °C (average site temperature) over 90 days confirmed that a notable decrease in hydrocarbon concentrations occurred in soils fractions with abundant bioaccessible pores and with a larger number of pores between 10 and 100 μm. The hydrocarbon degradation in bioactive soil fractions was extended to relatively high-molecular-weight hydrocarbons (C16-C34). This study provides quantitative information about how internal soil pore characteristics can influence bioremediation performance.

  11. Assessment of the 3 D Pore Structure and Individual Components of Preshaped Catalyst Bodies by X-Ray Imaging

    PubMed Central

    da Silva, Julio C; Mader, Kevin; Holler, Mirko; Haberthür, David; Diaz, Ana; Guizar-Sicairos, Manuel; Cheng, Wu-Cheng; Shu, Yuying; Raabe, Jörg; Menzel, Andreas; van Bokhoven, Jeroen A

    2015-01-01

    Porosity in catalyst particles is essential because it enables reactants to reach the active sites and it enables products to leave the catalyst. The engineering of composite-particle catalysts through the tuning of pore-size distribution and connectivity is hampered by the inability to visualize structure and porosity at critical-length scales. Herein, it is shown that the combination of phase-contrast X-ray microtomography and high-resolution ptychographic X-ray tomography allows the visualization and characterization of the interparticle pores at micro- and nanometer-length scales. Furthermore, individual components in preshaped catalyst bodies used in fluid catalytic cracking, one of the most used catalysts, could be visualized and identified. The distribution of pore sizes, as well as enclosed pores, which cannot be probed by traditional methods, such as nitrogen physisorption and isotherm analysis, were determined. PMID:26191088

  12. Organization of the Mitochondrial Apoptotic BAK Pore

    PubMed Central

    Aluvila, Sreevidya; Mandal, Tirtha; Hustedt, Eric; Fajer, Peter; Choe, Jun Yong; Oh, Kyoung Joon

    2014-01-01

    The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX “BH3-in-groove homodimer.” Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a “worm hole.” PMID:24337568

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

    SciTech Connect

    Hall, K.S.

    1986-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  15. 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.

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

    PubMed

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

    2016-01-01

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

  17. Magnetic-resonance pore imaging of nonsymmetric microscopic pore shapes

    NASA Astrophysics Data System (ADS)

    Hertel, Stefan Andreas; Wang, Xindi; Hosking, Peter; Simpson, M. Cather; Hunter, Mark; Galvosas, Petrik

    2015-07-01

    Imaging of the microstructure of porous media such as biological tissue or porous solids is of high interest in health science and technology, engineering and material science. Magnetic resonance pore imaging (MRPI) is a recent technique based on nuclear magnetic resonance (NMR) which allows us to acquire images of the average pore shape in a given sample. Here we provide details on the experimental design, challenges, and requirements of MRPI, including its calibration procedures. Utilizing a laser-machined phantom sample, we present images of microscopic pores with a hemiequilateral triangular shape even in the presence of NMR relaxation effects at the pore walls. We therefore show that MRPI is applicable to porous samples without a priori knowledge about their pore shape and symmetry. Furthermore, we introduce "MRPI mapping," which combines MRPI with conventional magnetic resonance imaging (MRI). This enables one to resolve microscopic pore sizes and shapes spatially, thus expanding the application of MRPI to samples with heterogeneous distributions of pores.

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2014-06-01

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

  20. Extreme accumulation of nucleotides in simulated hydrothermal pore systems

    PubMed Central

    Baaske, Philipp; Weinert, Franz M.; Duhr, Stefan; Lemke, Kono H.; Russell, Michael J.; Braun, Dieter

    2007-01-01

    We simulate molecular transport in elongated hydrothermal pore systems influenced by a thermal gradient. We find extreme accumulation of molecules in a wide variety of plugged pores. The mechanism is able to provide highly concentrated single nucleotides, suitable for operations of an RNA world at the origin of life. It is driven solely by the thermal gradient across a pore. On the one hand, the fluid is shuttled by thermal convection along the pore, whereas on the other hand, the molecules drift across the pore, driven by thermodiffusion. As a result, millimeter-sized pores accumulate even single nucleotides more than 108-fold into micrometer-sized regions. The enhanced concentration of molecules is found in the bulk water near the closed bottom end of the pore. Because the accumulation depends exponentially on the pore length and temperature difference, it is considerably robust with respect to changes in the cleft geometry and the molecular dimensions. Whereas thin pores can concentrate only long polynucleotides, thicker pores accumulate short and long polynucleotides equally well and allow various molecular compositions. This setting also provides a temperature oscillation, shown previously to exponentially replicate DNA in the protein-assisted PCR. Our results indicate that, for life to evolve, complicated active membrane transport is not required for the initial steps. We find that interlinked mineral pores in a thermal gradient provide a compelling high-concentration starting point for the molecular evolution of life. PMID:17494767

  1. Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation

    NASA Astrophysics Data System (ADS)

    Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W.; Lin, Jialing; Li, Jianing

    2016-07-01

    Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model — using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.

  2. Characterization of Two-pore Channel 2 (TPCN2)-mediated Ca2+ Currents in Isolated Lysosomes*

    PubMed Central

    Schieder, Michael; Rötzer, Katrin; Brüggemann, Andrea; Biel, Martin; Wahl-Schott, Christian A.

    2010-01-01

    Two-pore channels (TPCNs) have been proposed to form lysosomal Ca2+ release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca2+ channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca2+ selectivity. Our glass chip-based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels. PMID:20495006

  3. Characterization of two-pore channel 2 (TPCN2)-mediated Ca2+ currents in isolated lysosomes.

    PubMed

    Schieder, Michael; Rötzer, Katrin; Brüggemann, Andrea; Biel, Martin; Wahl-Schott, Christian A

    2010-07-01

    Two-pore channels (TPCNs) have been proposed to form lysosomal Ca(2+) release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca(2+) channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca(2+) selectivity. Our glass chip-based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels.

  4. Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation

    PubMed Central

    Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W.; Lin, Jialing; Li, Jianing

    2016-01-01

    Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model — using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation. PMID:27381287

  5. Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation.

    PubMed

    Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W; Lin, Jialing; Li, Jianing

    2016-01-01

    Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model - using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation. PMID:27381287

  6. Evaluation of Pore Networks in Caprocks at Geologic Storage Sites: A Combined Study using High Temperature and Pressure Reaction Experiments, Small Angle Neutron Scattering, and Focused Ion Beam-Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Mouzakis, K. M.; Sitchler, A.; Wang, X.; McCray, J. E.; Kaszuba, J. P.; Rother, G.; Dewers, T. A.; Heath, J. E.

    2011-12-01

    Low permeability rock units, often shales or mudstones, that overlie geologic formations under consideration for CO2 sequestration will help contain injected CO2. CO2 that does flow through these rocks will dissolve into the porewaters, creating carbonic acid lowering the pH. This perturbation of the system may result in mineral dissolution or precipitation, which can change the pore structure and impact the flow properties of the caprocks. In order to investigate the impacts that reaction can have on caprock pore structure, we performed a combination of high pressure high temperature reaction experiments, small angle neutron scattering (SANS) experiments and high resolution focused ion beam-scanning electron microscope (FIB-SEM) imaging on samples from the Gothic shale and Marine Tuscaloosa Group. Small angle neutron scattering was performed on unreacted and reacted caprocks at the High Flux Isotope Reactor at Oak Ridge National Laboratory. New precipitates and pores are observed in high-resolution images of the reacted samples. The precipitates have been preliminarily identified as gypsum or anhydrite, and sulfide minerals. Results from small angle neutron scattering, a technique that provides information about pores and pore/mineral interfaces at scales ~ 5 to 300 nm, show an increased porosity and specific surface area after reaction with brine and CO2. However, there appear to be differences in how the pore networks change between the two samples that are related to sample mineralogy and original pore network structure. Changes to pores and formation of new pores may lead to different capillary sealing behavior and permeability. This combination of controlled laboratory experiments, neutron scattering and high-resolution imaging provides detailed information about the geochemical processes that occur at the pore scale as CO2 reacts with rocks underground. Such information is integral to the evaluation of large-scale CO2 sequestration as a feasible technology

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. Decreasing transmembrane segment length greatly decreases perfringolysin O pore size

    SciTech Connect

    Lin, Qingqing; Li, Huilin; Wang, Tong; London, Erwin

    2015-04-08

    Perfringolysin O (PFO) is a transmembrane (TM) β-barrel protein that inserts into mammalian cell membranes. Once inserted into membranes, PFO assembles into pore-forming oligomers containing 30–50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores. In this study, we found that altering PFO TM segment length can alter the size of PFO pores. A PFO mutant with lengthened TM segments oligomerized to a similar extent as wild-type PFO, and exhibited pore-forming activity and a pore size very similar to wild-type PFO as measured by electron microscopy and a leakage assay. In contrast, PFO with shortened TM segments exhibited a large reduction in pore-forming activity and pore size. This suggests that the interaction between TM segments can greatly affect the size of pores formed by TM β-barrel proteins. PFO may be a promising candidate for engineering pore size for various applications.

  13. Decreasing transmembrane segment length greatly decreases perfringolysin O pore size

    DOE PAGES

    Lin, Qingqing; Li, Huilin; Wang, Tong; London, Erwin

    2015-04-08

    Perfringolysin O (PFO) is a transmembrane (TM) β-barrel protein that inserts into mammalian cell membranes. Once inserted into membranes, PFO assembles into pore-forming oligomers containing 30–50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores. In this study, we found that altering PFO TM segment length can alter the size of PFO pores. A PFO mutant with lengthened TM segments oligomerized to a similar extent as wild-type PFO, and exhibited pore-forming activity and a pore size very similar to wild-type PFO as measured by electron microscopy and a leakagemore » assay. In contrast, PFO with shortened TM segments exhibited a large reduction in pore-forming activity and pore size. This suggests that the interaction between TM segments can greatly affect the size of pores formed by TM β-barrel proteins. PFO may be a promising candidate for engineering pore size for various applications.« less

  14. 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.

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

    SciTech Connect

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

    1993-12-01

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

  16. 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.

  17. 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

  18. Pore pressure embrittlement in a volcanic edifice

    NASA Astrophysics Data System (ADS)

    Farquharson, Jamie; Heap, Michael J.; Baud, Patrick; Reuschlé, Thierry; Varley, Nick R.

    2016-01-01

    conduit, potentially explaining commonly observed near-conduit outgassing at many active volcanoes. Further, rock embrittlement at depth may create transient outgassing pathways by linking fracture networks near the edifice to larger-scale regional fault systems. Our experimental results affirm that pore pressure fluctuations associated with volcanic unrest may play a crucial role in dictating the evolution of a volcanic system.

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

    PubMed

    Hiyamuta, S; Ito, K

    1994-04-01

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

  20. 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.

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

    SciTech Connect

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

    1993-12-01

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

  2. A lipocentric view of peptide-induced pores.

    PubMed

    Fuertes, Gustavo; Giménez, Diana; Esteban-Martín, Santi; Sánchez-Muñoz, Orlando L; Salgado, Jesús

    2011-04-01

    Although lipid membranes serve as effective sealing barriers for the passage of most polar solutes, nonmediated leakage is not completely improbable. A high activation energy normally keeps unassisted bilayer permeation at a very low frequency, but lipids are able to self-organize as pores even in peptide-free and protein-free membranes. The probability of leakage phenomena increases under conditions such as phase coexistence, external stress or perturbation associated to binding of nonlipidic molecules. Here, we argue that pore formation can be viewed as an intrinsic property of lipid bilayers, with strong similarities in the structure and mechanism between pores formed with participation of peptides, lipidic pores induced by different types of stress, and spontaneous transient bilayer defects driven by thermal fluctuations. Within such a lipocentric framework, amphipathic peptides are best described as pore-inducing rather than pore-forming elements. Active peptides bound to membranes can be understood as a source of internal surface tension which facilitates pore formation by diminishing the high activation energy barrier. This first or immediate action of the peptide has some resemblance to catalysis. However, the presence of membrane-active peptides has the additional effect of displacing the equilibrium towards the pore-open state, which is then maintained over long times, and reducing the size of initial individual pores. Thus, pore-inducing peptides, regardless of their sequence and oligomeric organization, can be assigned a double role of increasing the probability of pore formation in membranes to high levels as well as stabilizing these pores after they appear.

  3. The Arabidopsis Nuclear Pore and Nuclear Envelope

    PubMed Central

    Meier, Iris; Brkljacic, Jelena

    2010-01-01

    The nuclear envelope is a double membrane structure that separates the eukaryotic cytoplasm from the nucleoplasm. The nuclear pores embedded in the nuclear envelope are the sole gateways for macromolecular trafficking in and out of the nucleus. The nuclear pore complexes assembled at the nuclear pores are large protein conglomerates composed of multiple units of about 30 different nucleoporins. Proteins and RNAs traffic through the nuclear pore complexes, enabled by the interacting activities of nuclear transport receptors, nucleoporins, and elements of the Ran GTPase cycle. In addition to directional and possibly selective protein and RNA nuclear import and export, the nuclear pore gains increasing prominence as a spatial organizer of cellular processes, such as sumoylation and desumoylation. Individual nucleoporins and whole nuclear pore subcomplexes traffic to specific mitotic locations and have mitotic functions, for example at the kinetochores, in spindle assembly, and in conjunction with the checkpoints. Mutants of nucleoporin genes and genes of nuclear transport components lead to a wide array of defects from human diseases to compromised plant defense responses. The nuclear envelope acts as a repository of calcium, and its inner membrane is populated by functionally unique proteins connected to both chromatin and—through the nuclear envelope lumen—the cytoplasmic cytoskeleton. Plant nuclear pore and nuclear envelope research—predominantly focusing on Arabidopsis as a model—is discovering both similarities and surprisingly unique aspects compared to the more mature model systems. This chapter gives an overview of our current knowledge in the field and of exciting areas awaiting further exploration. PMID:22303264

  4. Pore dynamics in lipid membranes

    NASA Astrophysics Data System (ADS)

    Gozen, I.; Dommersnes, P.

    2014-09-01

    Transient circular pores can open in plasma membrane of cells due to mechanical stress, and failure to repair such pores lead to cell death. Similar pores in the form of defects also exist among smectic membranes, such as in myelin sheaths or mitochondrial membranes. The formation and growth of membrane defects are associated with diseases, for example multiple sclerosis. A deeper understanding of membrane pore dynamics can provide a more refined picture of membrane integrity-related disease development, and possibly also treatment options and strategies. Pore dynamics is also of great importance regarding healthcare applications such as drug delivery, gene or as recently been implied, cancer therapy. The dynamics of pores significantly differ in stacks which are confined in 2D compared to those in cells or vesicles. In this short review, we will summarize the dynamics of different types of pores that can be observed in biological membranes, which include circular transient, fusion and hemi-fusion pores. We will dedicate a section to floral and fractal pores which were discovered a few years ago and have highly peculiar characteristics. Finally, we will discuss the repair mechanisms of large area pores in conjunction with the current cell membrane repair hypotheses.

  5. Change of pore properties during carbonization of coking coal

    SciTech Connect

    Miura, S.; Silveston, P.L.

    1980-01-01

    Porosimetry, sorption and density measurements are reported on two caking bituminous coals. West Virginia Jewel No. 2 medium volatile and a Pennsylvania Pittsburgh seam high volatile C, for final carbonization temperatures between 400 and 1000/sup 0/C. Samples were not confined and heating rates of 3 and 8.2%/min were employed. The medium volatile samples exhibit pronounced maxima in pore volume, pore surface area and porosity between 600 and 800/sup 0/C. These temperatures are unexpectedly greater than those at which maximum particle dilation and maximum rate of devolatilization occur. An explanation for this observation is that closed pores are created during carbonization below 600/sup 0/C which are opened when carbonization is carried to higher temperatures. Shape of the pore volume curves suggest new pore initiation, pore growth and pore shrinkage are the dominant processes operating, although collapse of the ultra micro pore structure seems to occur above 800/sup 0/C. A pore development model employing simple expressions for the three dominant processes successfully predicts the pore volume and surface area changes. Apparent activation energies derived from the model indicate that the rates of these dominant steps are controled by basicaly physical, not chemical, changes.

  6. The nuclear pore complex and nuclear transport.

    PubMed

    Wente, Susan R; Rout, Michael P

    2010-10-01

    Internal membrane bound structures sequester all genetic material in eukaryotic cells. The most prominent of these structures is the nucleus, which is bounded by a double membrane termed the nuclear envelope (NE). Though this NE separates the nucleoplasm and genetic material within the nucleus from the surrounding cytoplasm, it is studded throughout with portals called nuclear pore complexes (NPCs). The NPC is a highly selective, bidirectional transporter for a tremendous range of protein and ribonucleoprotein cargoes. All the while the NPC must prevent the passage of nonspecific macromolecules, yet allow the free diffusion of water, sugars, and ions. These many types of nuclear transport are regulated at multiple stages, and the NPC carries binding sites for many of the proteins that modulate and modify the cargoes as they pass across the NE. Assembly, maintenance, and repair of the NPC must somehow occur while maintaining the integrity of the NE. Finally, the NPC appears to be an anchor for localization of many nuclear processes, including gene activation and cell cycle regulation. All these requirements demonstrate the complex design of the NPC and the integral role it plays in key cellular processes. PMID:20630994

  7. Analysis of a spatially deconvolved solar pore

    NASA Astrophysics Data System (ADS)

    Quintero Noda, C.; Shimizu, T.; Ruiz Cobo, B.; Suematsu, Y.; Katsukawa, Y.; Ichimoto, K.

    2016-08-01

    Solar pores are active regions with large magnetic field strengths and apparent simple magnetic configurations. Their properties resemble the ones found for the sunspot umbra although pores do not show penumbra. Therefore, solar pores present themselves as an intriguing phenomenon that is not completely understood. We examine in this work a solar pore observed with Hinode/SP using two state of the art techniques. The first one is the spatial deconvolution of the spectropolarimetric data that allows removing the stray light contamination induced by the spatial point spread function of the telescope. The second one is the inversion of the Stokes profiles assuming local thermodynamic equilibrium that let us to infer the atmospheric physical parameters. After applying these techniques, we found that the spatial deconvolution method does not introduce artefacts, even at the edges of the magnetic structure, where large horizontal gradients are detected on the atmospheric parameters. Moreover, we also describe the physical properties of the magnetic structure at different heights finding that, in the inner part of the solar pore, the temperature is lower than outside, the magnetic field strength is larger than 2 kG and unipolar, and the line-of-sight velocity is almost null. At neighbouring pixels, we found low magnetic field strengths of same polarity and strong downward motions that only occur at the low photosphere, below the continuum optical depth log τ = -1. Finally, we studied the spatial relation between different atmospheric parameters at different heights corroborating the physical properties described before.

  8. Wrinkles and Folds of Activated Graphene Nanosheets as Fast and Efficient Adsorptive Sites for Hydrophobic Organic Contaminants.

    PubMed

    Wang, Jun; Chen, Baoliang; Xing, Baoshan

    2016-04-01

    To create more wrinkles and folds as available adsorption sites, graphene nanosheets (GNS) were thermally treated with KOH for morphological alteration. The surface structures and properties of the activated graphene nanosheets (AGN) were characterized by BET-N2, SEM, TEM, Raman, XRD, XPS, and FTIR. After KOH etching, the highly crystal structure was altered, self-aggregation of graphene layers were evidently relieved, and more single to few layer graphene nanosheets were created with wrinkles and folds. Also both specific surface area and micropore volume of AGN increased relative to GNS. The adsorption of AGN toward p-nitrotoluene, naphthalene and phenanthrene were greatly enhanced in comparison with GNS, and gradually promoted with increasing degree of KOH etching. Adsorption rate of organic contaminants on AGN was very fast and efficient, whereas small molecules showed higher adsorption rates due to the more porous surface of graphene. In addition to π-π interaction, the high affinities of p-nitrotoluene to AGN are suggested from strong electron charge transfer interactions between nitro groups on p-nitrotoluene and defect sites of AGN. A positively linear correlation between organic molecule uptake and the micropore volume of AGN indicated that pore-filling mechanism may play an important role in adsorption. Morphological wrinkles and folds of graphene nanosheets can be regulated to enhance the adsorption capability and kinetics for efficient pollutant removal and to selectively preconcentrate adsorbates with different sizes for detection.

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

    SciTech Connect

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

    2010-09-07

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

  10. The structure of a melittin-stabilized pore.

    PubMed

    Leveritt, John M; Pino-Angeles, Almudena; Lazaridis, Themis

    2015-05-19

    Melittin has been reported to form toroidal pores under certain conditions, but the atomic-resolution structure of these pores is unknown. A 9-μs all-atom molecular-dynamics simulation starting from a closely packed transmembrane melittin tetramer in DMPC shows formation of a toroidal pore after 1 μs. The pore remains stable with a roughly constant radius for the rest of the simulation. Surprisingly, one or two melittin monomers frequently transition between transmembrane and surface states. All four peptides are largely helical. A simulation in a DMPC/DMPG membrane did not lead to a stable pore, consistent with the experimentally observed lower activity of melittin on anionic membranes. The picture that emerges from this work is rather close to the classical toroidal pore, but more dynamic with respect to the configuration of the peptides. PMID:25992720

  11. Enzyme screening with synthetic multifunctional pores: Focus on biopolymers

    PubMed Central

    Sordé, Nathalie; Das, Gopal; Matile, Stefan

    2003-01-01

    This report demonstrates that a single set of identical synthetic multifunctional pores can detect the activity of many different enzymes. Enzymes catalyzing either synthesis or degradation of DNA (exonuclease III or polymerase I), RNA (RNase A), polysaccharides (heparinase I, hyaluronidase, and galactosyltransferase), and proteins (papain, ficin, elastase, subtilisin, and pronase) are selected to exemplify this key characteristic of synthetic multifunctional pore sensors. Because anionic, cationic, and neutral substrates can gain access to the interior of complementarily functionalized pores, such pores can be the basis for very user-friendly screening of a broad range of enzymes. PMID:14530413

  12. 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

  13. How realistic is the pore size distribution calculated from adsorption isotherms if activated carbon is composed of fullerene-like fragments?

    PubMed

    Terzyk, Artur P; Furmaniak, Sylwester; Harris, Peter J F; Gauden, Piotr A; Włoch, Jerzy; Kowalczyk, Piotr; Rychlicki, Gerhard

    2007-11-28

    A plausible model for the structure of non-graphitizing carbon is one which consists of curved, fullerene-like fragments grouped together in a random arrangement. Although this model was proposed several years ago, there have been no attempts to calculate the properties of such a structure. Here, we determine the density, pore size distribution and adsorption properties of a model porous carbon constructed from fullerene-like elements. Using the method proposed recently by Bhattacharya and Gubbins (BG), which was tested in this study for ideal and defective carbon slits, the pore size distributions (PSDs) of the initial model and two related carbon models are calculated. The obtained PSD curves show that two structures are micro-mesoporous (with different ratio of micro/mesopores) and the third is strictly microporous. Using the grand canonical Monte Carlo (GCMC) method, adsorption isotherms of Ar (87 K) are simulated for all the structures. Finally PSD curves are calculated using the Horvath-Kawazoe, non-local density functional theory (NLDFT), Nguyen and Do, and Barrett-Joyner-Halenda (BJH) approaches, and compared with those predicted by the BG method. This is the first study in which different methods of calculation of PSDs for carbons from adsorption data can be really verified, since absolute (i.e. true) PSDs are obtained using the BG method. This is also the first study reporting the results of computer simulations of adsorption on fullerene-like carbon models.

  14. Latent fingermark pore area reproducibility.

    PubMed

    Gupta, A; Buckley, K; Sutton, R

    2008-08-01

    The study of the reproducibility of friction ridge pore detail in fingermarks is a measure of their usefulness in personal identification. Pore area in latent prints developed using cyanoacrylate and ninhydrin were examined and measured by photomicrography using appropriate software tools. The data were analysed statistically and the results showed that pore area is not reproducible in developed latent prints, using either of the development techniques. The results add further support to the lack of reliability of pore area in personal identification. PMID:18617339

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

    PubMed

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

    2014-05-21

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

  16. Hydrochromic Approaches to Mapping Human Sweat Pores.

    PubMed

    Park, Dong-Hoon; Park, Bum Jun; Kim, Jong-Man

    2016-06-21

    colorimetric change near body temperature. This feature enables the use of this technique to generate high-quality images of sweat pores. This Account also focuses on the results of the most recent phase of this investigation, which led to the development of a simple yet efficient and reliable technique for sweat pore mapping. The method utilizes a hydrophilic polymer composite film containing fluorescein, a commercially available dye that undergoes a fluorometric response as a result of water-dependent interconversion between its ring-closed spirolactone (nonfluorescent) and ring-opened fluorone (fluorescent) forms. Surface-modified carbon nanodots (CDs) have also been found to be efficient for hydrochromic mapping of human sweat pores. The results discovered by Lou et al. [ Adv. Mater. 2015 , 27 , 1389 ] are also included in this Account. Sweat pore maps obtained from fingertips using these materials were found to be useful for fingerprint analysis. In addition, this hydrochromism-based approach is sufficiently sensitive to enable differentiation between sweat-secreting active pores and inactive pores. As a result, the techniques can be applied to clinical diagnosis of malfunctioning sweat pores. The directions that future research in this area will follow are also discussed. PMID:27159417

  17. 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)

  18. Cilia and Nuclear Pore Proteins: Pore No More?

    PubMed

    Obado, Samson O; Rout, Michael P

    2016-09-12

    Nuclear pore proteins at the base of cilia were thought to regulate transport into cilia. In this issue of Developmental Cell, Del Viso et al. (2016) challenge this view, showing instead that pore proteins localize to ciliary basal bodies and that their perturbation leads to congenital heart disease. PMID:27623377

  19. 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.

  20. Blockers of VacA provide insights into the structure of the pore.

    PubMed Central

    Tombola, F; Del Giudice, G; Papini, E; Zoratti, M

    2000-01-01

    The cytotoxic effects of the Helicobacter pylori toxin VacA, an important etiogenic factor in human gastric diseases, are due to its ability to form anion-selective pores in target cell membranes. We have studied the inhibition of channel activity by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), representatives of two popular classes of chloride channel blockers, to gain information on the mechanism of blocking and on the unknown structure of the VacA pore. The data indicate that both compounds produce a fast block by binding to separate but mutually exclusive sites within the channel lumen. DIDS binds close to the pore opening on the side of protein insertion, whereas NPPB blocks at a position in the opposite half of the channel. Although DIDS reaches the blocking site by traveling along the lumen, inhibition by NPPB appears to involve mainly partition of the compound into the membrane, voltage-independent diffusion from it to the inhibitory position, and voltage-dependent exit. The data are consistent with a pore that can be more easily entered from the side of protein insertion than from the opposite end. PMID:10920018

  1. 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.

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

    PubMed Central

    Stec, Boguslaw

    2012-01-01

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

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

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-09-18

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

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

    PubMed Central

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

    2012-01-01

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

  5. Petrophysical characterization of first ever drilled core samples from an active CO2 storage site, the German Ketzin Pilot Site - Comparison with long term experiments

    NASA Astrophysics Data System (ADS)

    Zemke, Kornelia; Liebscher, Axel

    2014-05-01

    Petrophysical properties like porosity and permeability are key parameters for a safe long-term storage of CO2 but also for the injection operation itself. These parameters may change during and/or after the CO2 injection due to geochemical reactions in the reservoir system that are triggered by the injected CO2. Here we present petrophysical data of first ever drilled cores from a newly drilled well at the active CO2 storage site - the Ketzin pilot site in the Federal State of Brandenburg, Germany. By comparison with pre-injection baseline data from core samples recovered prior to injection, the new samples provide the unique opportunity to evaluate the impact of CO2 on pore size related properties of reservoir and cap rocks at a real injection site under in-situ reservoir conditions. After injection of 61 000 tons CO2, an additional well was drilled and new rock cores were recovered. In total 100 core samples from the reservoir and the overlaying caprock were investigated by NMR relaxation. Permeability of 20 core samples was estimated by nitrogen and porosity by helium pycnometry. The determined data are comparable between pre-injection and post-injection core samples. The lower part of the reservoir sandstone is unaffected by the injected CO2. The upper part of the reservoir sandstone shows consistently slightly lower NMR porosity and permeability values in the post-injection samples when compared to the pre-injection data. This upper sandstone part is above the fluid level and CO2 present as a free gas phase and a possible residual gas saturation of the cores distorted the NMR results. The potash-containing drilling fluid can also influence these results: NMR investigation of twin samples from inner and outer parts of the cores show a reduced fraction of larger pores for the outer core samples together with lower porosities and T2 times. The drill mud penetration depth can be controlled by the added fluorescent tracer. Due to the heterogeneous character of the

  6. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

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

    ERIC Educational Resources Information Center

    Heo, Gyeong Mi; Lee, Romee

    2013-01-01

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

  9. Two functionally distinct subsites for the binding of internal blockers to the pore of voltage-activated K+ channels

    PubMed Central

    Baukrowitz, Thomas; Yellen, Gary

    1996-01-01

    Many blockers of Na+ and K+ channels act by blocking the pore from the intracellular side. For Shaker K+ channels, such intracellular blockers vary in their functional effect on slow (C-type) inactivation: Some blockers interfere with C-type inactivation, whereas others do not. These functional differences can be explained by supposing that there are two overlapping “subsites” for blocker binding, only one of which inhibits C-type inactivation through an allosteric effect. We find that the ability to bind to these subsites depends on specific structural characteristics of the blockers, and correlates with the effect of mutations in two distinct regions of the channel protein. These interactions are important because they affect the ability of blockers to produce use-dependent inhibition. PMID:8917595

  10. 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.

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

    PubMed

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

    2015-12-29

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

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

    PubMed

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

    2015-03-19

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

  13. 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

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

    PubMed

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

    2015-07-13

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

  15. 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.

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

    PubMed

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

    2016-07-01

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

  17. Micro-CT scan reveals an unexpected high-volume and interconnected pore network in a Cretaceous Sanagasta dinosaur eggshell.

    PubMed

    Hechenleitner, E Martín; Grellet-Tinner, Gerald; Foley, Matthew; Fiorelli, Lucas E; Thompson, Michael B

    2016-03-01

    The Cretaceous Sanagasta neosauropod nesting site (La Rioja, Argentina) was the first confirmed instance of extinct dinosaurs using geothermal-generated heat to incubate their eggs. The nesting strategy and hydrothermal activities at this site led to the conclusion that the surprisingly 7 mm thick-shelled eggs were adapted to harsh hydrothermal microenvironments. We used micro-CT scans in this study to obtain the first three-dimensional microcharacterization of these eggshells. Micro-CT-based analyses provide a robust assessment of gas conductance in fossil dinosaur eggshells with complex pore canal systems, allowing calculation, for the first time, of the shell conductance through its thickness. This novel approach suggests that the shell conductance could have risen during incubation to seven times more than previously estimated as the eggshell erodes. In addition, micro-CT observations reveal that the constant widening and branching of pore canals form a complex funnel-like pore canal system. Furthermore, the high density of pore canals and the presence of a lateral canal network in the shell reduce the risks of pore obstruction during the extended incubation of these eggs in a relatively highly humid and muddy nesting environment. PMID:27009182

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

    PubMed

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

    1995-10-01

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

  19. 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.

  20. 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

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

  2. Field Summary Report for Remedial Investigation of Hanford Site Releases to the Columbia River, Hanford Site, Washington

    SciTech Connect

    L.C. Hulstrom

    2010-08-11

    This report summarizes field sampling activities conducted in support of WCH’s Remedial Investigation of Hanford Site Releases to the Columbia River. This work was conducted form 2008 through 2010. The work included preliminary mapping and measurement of Hanford Site contaminants in sediment, pore water, and surface water located in areas where groundwater upwelling were found.

  3. Field Summary Report for Remedial Investigation of Hanford Site Releases to the Coumbia River, Hanford Site, Washington

    SciTech Connect

    L.C. Hulstrom

    2010-11-10

    This report summarizes field sampling activities conducted in support of WCH’s Remedial Investigation of Hanford Site Releases to the Columbia River. This work was conducted form 2008 through 2010. The work included preliminary mapping and measurement of Hanford Site contaminants in sediment, pore water, and surface water located in areas where groundwater upwelling were found.

  4. 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.

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

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

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

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

    PubMed

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

    2006-11-01

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

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

    PubMed

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

    2014-12-12

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

  8. 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…

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

    PubMed

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

    2016-09-12

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

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

    PubMed

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

    2013-01-01

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

  11. 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.

  12. 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.

  13. 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

  14. Process of inducing pores in membranes by melittin

    PubMed Central

    Lee, Ming-Tao; Sun, Tzu-Lin; Hung, Wei-Chin; Huang, Huey W.

    2013-01-01

    Melittin is a prototype of the ubiquitous antimicrobial peptides that induce pores in membranes. It is commonly used as a molecular device for membrane permeabilization. Even at concentrations in the nanomolar range, melittin can induce transient pores that allow transmembrane conduction of atomic ions but not leakage of glucose or larger molecules. At micromolar concentrations, melittin induces stable pores allowing transmembrane leakage of molecules up to tens of kilodaltons, corresponding to its antimicrobial activities. Despite extensive studies, aspects of the molecular mechanism for pore formation remain unclear. To clarify the mechanism, one must know the states of the melittin-bound membrane before and after the process. By correlating experiments using giant unilamellar vesicles with those of peptide-lipid multilayers, we found that melittin bound on the vesicle translocated and redistributed to both sides of the membrane before the formation of stable pores. Furthermore, stable pores are formed only above a critical peptide-to-lipid ratio. The initial states for transient and stable pores are different, which implies different mechanisms at low and high peptide concentrations. To determine the lipidic structure of the pore, the pores in peptide–lipid multilayers were induced to form a lattice and examined by anomalous X-ray diffraction. The electron density distribution of lipid labels shows that the pore is formed by merging of two interfaces through a hole. The molecular property of melittin is such that it adsorbs strongly to the bilayer interface. Pore formation can be viewed as the bilayer adopting a lipid configuration to accommodate its excessive interfacial area. PMID:23940362

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

    PubMed Central

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

    2015-01-01

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

  16. A mutation in the pore of the sodium channel alters gating.

    PubMed Central

    Tomaselli, G F; Chiamvimonvat, N; Nuss, H B; Balser, J R; Pérez-García, M T; Xu, R H; Orias, D W; Backx, P H; Marban, E

    1995-01-01

    Ion permeation and channel gating are classically considered independent processes, but site-specific mutagenesis studies in K channels suggest that residues in or near the ion-selective pore of the channel can influence activation and inactivation. We describe a mutation in the pore of the skeletal muscle Na channel that alters gating. This mutation, I-W53C (residue 402 in the mu 1 sequence), decreases the sensitivity to block by tetrodotoxin and increases the sensitivity to block by externally applied Cd2+ relative to the wild-type channel, placing this residue within the pore near the external mouth. Based on contemporary models of the structure of the channel, this residue is remote from the regions of the channel known to be involved in gating, yet this mutation abbreviates the time to peak and accelerates the decay of the macroscopic Na current. At the single-channel level we observe a shortening of the latency to first opening and a reduction in the mean open time compared with the wild-type channel. The acceleration of macroscopic current kinetics in the mutant channels can be simulated by changing only the activation and deactivation rate constants while constraining the microscopic inactivation rate constants to the values used to fit the wild-type currents. We conclude that the tryptophan at position 53 in the domain IP-loop may act as a linchpin in the pore that limits the opening transition rate. This effect could reflect an interaction of I-W53 with the activation voltage sensors or a more global gating-induced change in pore structure. Images FIGURE 1 PMID:7612823

  17. Open–closed switching of synthetic tubular pores

    PubMed Central

    Kim, Yongju; Kang, Jiheong; Shen, Bowen; Wang, Yanqiu; He, Ying; Lee, Myongsoo

    2015-01-01

    While encouraging progress has been made on switchable nanopores to mimic biological channels and pores, it remains a great challenge to realize long tubular pores with a dynamic open–closed motion. Here we report μm-long, dynamic tubular pores that undergo rapid switching between open and closed states in response to a thermal signal in water. The tubular walls consist of laterally associated primary fibrils stacked from disc-shaped molecules in which the discs readily tilt by means of thermally regulated dehydration of the oligoether chains placed on the wall surfaces. Notably, this pore switching mediates a controlled water-pumping catalytic action for the dehydrative cyclization of adenosine monophosphate to produce metabolically active cyclic adenosine monophosphate. We believe that our work may allow the creation of a variety of dynamic pore structures with complex functions arising from open–closed motion. PMID:26456695

  18. Metal structures with parallel pores

    NASA Technical Reports Server (NTRS)

    Sherfey, J. M.

    1976-01-01

    Four methods of fabricating metal plates having uniformly sized parallel pores are studied: elongate bundle, wind and sinter, extrude and sinter, and corrugate stack. Such plates are suitable for electrodes for electrochemical and fuel cells.

  19. Pore-forming activity of Coxiella burnetii outer membrane protein oligomer comprised of 29.5- and 31-kDa polypeptides. Inhibition of porin activity by monoclonal antibodies 4E8 and 4D6.

    PubMed

    Banerjee-Bhatnagar, N; Bolt, C R; Williams, J C

    1996-07-23

    Envelopes of large-cell variant Coxiella burnetii, the agent of Q fever, were the starting material for purification of an outer membrane protein (OMP) oligomer with aggregate molecular mass of approximately 2 x 10(4) kDa. The oligomer was resistant to trypsin and dissociation by SDS at 100 degrees C. Reducing agents dissociated the oligomer into monomers of 29.5 and 31 kDa, which migrated as a doublet during SDS-polyacrylamide gel electrophoresis. Both monomers were reactive in an immunoblot assay with monoclonal antibodies (mAbs) 4E8 and 4D6, which were previously selected for their reactivity with purified and SDS-denatured 29.5 kDa protein. Proteoliposomes were functional in an equilibrium assay at pH 7 and a swelling assay at pH 7 and 4.5. The pores in proteoliposomes allowed the passage of arabinose, glucose, and sucrose, but restricted stachyose. Polyclonal antibodies to C. burnetii cells and the mAbs were able to bind C. burnetii at pH 7 and 4.5. The uptake of 14C-glucose at pH 4.5 was inhibited by polyclonal antibodies and mAbs after binding to cells at pH 7. The mAbs did not inhibit 14C-glucose uptake at pH 4.5 after binding to cells at pH 4.5. Although the mAbs bind C. burnetii porin epitopes before and after acid activation, the mAbs bound under acidic conditions were unable to inhibit porin function. The inhibition of porin channel function by mAbs confirms the role of porin as a permeability barrier for the subsequent active transport of glucose by C. burnetii. In another study, we showed that the 29.5 kDa OMP antigen induced active immunity against virulent challenge. This information, combined with the recent confirmation that porins are important antigens in the induction of specific protective immune responses against infection by gram-negative bacteria, suggests that humoral immunity directed against C. burnetii porins might play an important role in immunity against Q fever (human infection) and coxiellosis (animal infection), global enzootic

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

    PubMed

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

    2015-05-20

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

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

    PubMed

    Muramatsu, Akiyoshi

    2016-01-01

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

  2. Antimicrobial peptides bind more strongly to membrane pores

    PubMed Central

    Mihajlovic, Maja

    2010-01-01

    Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize bacterial membranes. Understanding their mechanism of action might help design better antibiotics. Using an implicit membrane model, modified to include pores of different shapes, we show that four AMPs (alamethicin, melittin, a magainin analogue, MG-H2, and piscidin 1) bind more strongly to membrane pores, consistent with the idea that they stabilize them. The effective energy of alamethicin in cylindrical pores is similar to that in toroidal pores, whereas the effective energy of the other three peptides is lower in toroidal pores. Only alamethicin intercalates into the membrane core; MG-H2, melittin and piscidin are located exclusively at the hydrophobic/hydrophilic interface. In toroidal pores, the latter three peptides often bind at the edge of the pore, and are in an oblique orientation. The calculated binding energies of the peptides are correlated with their hemolytic activities. We hypothesize that one distinguishing feature of AMPs may be the fact that they are imperfectly amphipathic which allows them to bind more strongly to toroidal pores. An initial test on a melittin-based mutant seems to support this hypothesis. PMID:20188066

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

    PubMed

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

    2015-05-29

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

  4. Rates of silicate dissolution in deep-sea sediment: In situ measurement using 234U/238U of pore fluids

    SciTech Connect

    Maher, Katharine; DePaolo, Donald J.; Lin, Jo Chiu-Fang

    2004-11-22

    Bulk dissolution rates for sediment from ODP Site 984A in the North Atlantic are determined using the 234U/238U activity ratios of pore water, bulk sediment, and leachates. Site 984A is one of only several sites where closely spaced pore water samples were obtained from the upper 60 meters of the core; the sedimentation rate is high (11 15 cm/ka), hence the sediments in the upper 60 meters are less than 500 ka old. The sediment is clayey silt and composed mostly of detritus derived from Iceland with a significant component of biogenic carbonate (up to 30 percent).The pore water 234U/238U activity ratios are higher than seawater values, in the range of 1.2 to 1.6, while the bulk sediment 234U/238U activity ratios are close to 1.0. The 234U/238U of the pore water reflects a balance between the mineral dissolution rate and the supply rate of excess 234U to the pore fluid by a-recoil injection of 234Th. The fraction of 238U decays that result in a-recoil injection of 234U to pore fluid is estimated to be 0.10 to 0.20 based on the 234U/238U of insoluble residue fractions. The calculated bulk dissolution rates, in units of g/g/yr are in the range of 4 x 10-7 to 2 x 10-6 yr-1. There is significant down-hole variability in pore water 234U/238U activity ratios (and hence dissolution rates) on a scale of ca. 10 m. The inferred bulk dissolution rate constants are 100 to 104 times slower than laboratory-determined rates, 100 times faster than rates inferred for older sediments based on Sr isotopes, and similar to weathering rates determined for terrestrial soils of similar age. The results of this study suggest that U isotopes can be used to measure in situ dissolution rates in fine-grained clastic materials. The rate estimates for sediments from ODP Site 984 confirm the strong dependence of reactivity on the age of the solid material: the bulk dissolution rate (Rd) of soils and deep-sea sediments can be approximately described by the expression Rd >> 0.1 Age-1 for ages

  5. Optimization of a pressurization methodology for extracting pore-water.

    PubMed

    Lopes, Isabel; Ribeiro, Rui

    2005-12-01

    Sediment toxicity can be assessed by conducting pore-water toxicity assays with standard water column organisms. Several methods have been developed for sampling pore-water. Centrifugation and pressurization methods are recommended when large volumes of pore-water are required to perform toxicity assays. Nevertheless, these methods involve sediment transportation and storage in laboratory, which can alter sediment toxicity. Therefore, an extraction method for large volumes that could be employed in the field site would be highly desirable. This study aimed to optimize and further evaluate an existing sediment pressurizing device with low construction costs, easy to carry and operate in the field, and presenting minimal chemical reactivity. The latter characteristic was achieved by lining the device interior with Teflon, by using large pore filters (50 microm), and by using an inert gas (nitrogen). Pore-water extraction efficiency and the toxicities of pore-water samples obtained by pressurization and by refrigerated centrifugation were compared. An artificial sediment (70% sand, 20% kaolin and 10% alpha-cellulose) spiked with an alcohol (phenol), a surfactant (SDS), a metal (copper), an organophosphate pesticide (parathion), and a natural sediment contaminated with acid mine drainage, were assayed for toxicity using Microtox assays. Sediment pressurization was found to be as efficient to extract pore-water as centrifugation, being more cost effective and adequate for field use.

  6. Assessment of metal toxicity in sediment pore water from Lake Macquarie, Australia.

    PubMed

    Doyle, C J; Pablo, F; Lim, R P; Hyne, R V

    2003-04-01

    Recent investigations into the level of heavy metal enrichment in the sediments of Lake Macquarie have indicated that significant contamination has occurred over the past 100 years, with elevated levels of lead, zinc, cadmium, copper, and selenium being observed in most parts of the lake. Pore water extracted from sediments showing the greatest contamination by these metals exhibited toxicity to the larval development of the sea urchin Heliocidaris tuberculata. However, an analysis of pore water metal concentrations revealed that the concentrations of these metals were too low to cause toxicity. Rather, pore water toxicity was highly correlated with manganese for the majority of sites sampled; subsequent spiking experiments confirmed manganese as a cause of toxicity. Current levels of manganese in the sediments of Lake Macquarie have arisen from natural sources and are not the result of anthropogenic activities. These results reiterate the importance of identifying the causes of toxicity in assessments of sediment contamination, particularly when testing sediment pore waters using sensitive early life stages. PMID:12712294

  7. Linker Installation: Engineering Pore Environment with Precisely Placed Functionalities in Zirconium MOFs.

    PubMed

    Yuan, Shuai; Chen, Ying-Pin; Qin, Jun-Sheng; Lu, Weigang; Zou, Lanfang; Zhang, Qiang; Wang, Xuan; Sun, Xing; Zhou, Hong-Cai

    2016-07-20

    Precise placement of multiple functional groups in a highly ordered metal-organic framework (MOF) platform allows the tailoring of the pore environment, which is required for advanced applications. To realize this, we present a comprehensive study on the linker installation method, in which a stable MOF with coordinatively unsaturated Zr6 clusters was employed and linkers bearing different functional groups were postsynthetically installed. A Zr-MOF with inherent missing linker sites, namely, PCN-700, was initially constructed under kinetic control. Twelve linkers with different substituents were then designed to study their effect on MOF formation kinetics and therefore resulting MOF structures. Guided by the geometrical analysis, linkers with different lengths were installed into a parent PCN-700, giving rise to 11 new MOFs and each bearing up to three different functional groups in predefined positions. Systematic variation of the pore volume and decoration of pore environment were realized by linker installation, which resulted in synergistic effects including an enhancement of H2 adsorption capacities of up to 57%. In addition, a size-selective catalytic system for aerobic alcohol oxidation reaction is built in PCN-700 through linker installation, which shows high activity and tunable size selectivity. Altogether, these results exemplify the capability of the linker installation method in the pore environment engineering of stable MOFs with multiple functional groups, giving an unparalleled level of control. PMID:27345035

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

    PubMed

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

    2015-03-01

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

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

    PubMed

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

    2015-03-01

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

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

    SciTech Connect

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

    1995-12-31

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

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

    SciTech Connect

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

    1992-11-01

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

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

    SciTech Connect

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

    1992-01-01

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

  13. 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...

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  15. 77 FR 5560 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

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

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

    NASA Astrophysics Data System (ADS)

    Qi, Jian-Xun; Jiang, Fan

    2011-05-01

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

  17. Soluble reactive phosphorus transport and retention in tropical, rainforest streams draining a volcanic and geothermally active landscape in Costa Rica.: Long-term concentration patterns, pore water environment and response to ENSO events

    USGS Publications Warehouse

    Triska, F.J.; Pringle, C.M.; Duff, J.H.; Avanzino, R.J.; Ramirez, A.; Ardon, M.; Jackman, A.P.

    2006-01-01

    Soluble reactive phosphorus (SRP) transport/retention was determined at four sites in three rainforest streams draining La Selva Biological Station, Costa Rica. La Selva is located at the base of the last remaining intact rainforest transect from 30 m above sea level to 3000 m along the entire Caribbean slope of Central America. Steam SRP levels can be naturally high there due to regional, geothermal groundwater discharged at ambient temperature. Monitoring since 1988 has revealed distinctive long-term differences in background SRP and total P (TP) for three streams in close proximity, and identified the impact of ENSO (El Nino Southern Oscillation) events on SRP-enriched reaches. Mean interannual SRP concentrations (?? standard deviation) were 89 ?? 53??g/l in the Salto (1988-1996), 21 ?? 39??g/l in the Pantano (1988-1998), and 26 ?? 35??g/l in the Sabalo (1988-1996). After January, 1997 the separate upland-lowland contributions to discharge and SRP load were determined monthly in the Salto. SRP in Upper Salto was low (19 ?? 8??g/l, 1997-2002) until enriched at the upland-lowland transition by regional groundwater. Mean SRP concentration in Lower Salto (108 ?? 104??g/l) was typically highest February-April, the driest months, and lowest July-September, the wettest. SRP concentration was positively correlated to the inverse of discharge in Lower Salto when ENSO data were omitted (1992 and 1998-1999), but not in the Upper Salto, Pantano, or Sabalo. TP was positively correlated to the inverse of discharge in all three streams when ENSO data were omitted. High SRP springs and seeps along the Lower Salto contributed 36% of discharge but 85% of SRP export 1997-2001. Annual SRP flux from the total Salto watershed (1997-2001) averaged 2.9 kg/ha year, but only 0.6 kg/ha year from the Upper Salto. A dye tracer injection showed that pore water environments were distinctly different between Upper and Lower Salto. Upper Salto had high surface water-pore water exchange, high

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

    PubMed

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

    2007-12-01

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

  19. 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.

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

    PubMed Central

    2015-01-01

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

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

    PubMed

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

    2013-12-01

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

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

    PubMed

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

    2014-04-01

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

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

    SciTech Connect

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

    1988-10-18

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

  4. 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.

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

    NASA Astrophysics Data System (ADS)

    Miyamoto, Daisuke; Ichikuni, Nobuyuki; Shimazu, Shogo

    2007-02-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

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

    1991-11-01

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

  8. Active surface faulting or landsliding in the Lower Tagus Valley (Portugal)? A solved controversy concerning the Vila Chã de Ourique site

    NASA Astrophysics Data System (ADS)

    Cabral, João Manuel; Marques, Fernando; Figueiredo, Paula; Matias, Luís

    2011-04-01

    The Lower Tagus Valley has experienced significant (M 6-7) historical seismicity, evidencing the presence of seismogenic faults. These are still deficiently known due to the low strain rates and the recent alluvial sedimentation of the Tagus River that buries most of the structures, though Paleoseismic evidence was allegedly found by a research team in the Tagus valley, at a site 60 km N of Lisbon, near Vila Chã de Ourique (VCO). According to this team, trenching at the VCO site exposed an active thrust fault, evidencing the surface rupture of a large earthquake that occurred in 1531. Our studies performed at this site, comprising field observations with a reappraisal of the trench outcrops previously excavated, borehole drilling, soil mechanics laboratory testing, and seismic reflection acquisition, pointed to the alternative interpretation that the outcropping structures are gravitational and not of tectonic origin. The interpretation of new outcrops crosscutting the structures exposed at the trenches, as well as newly acquired high-resolution seismic reflection data, definitely exclude the active thrust fault explanation and support a gravitational slip model for all the observed structures. Gravitational slip in the river bank slope was promoted by low shear strength clays and high pore water pressure coupled with slope toe river erosion. Gravitational slides must have occurred prior to development of the present sedimentation level of the Tagus alluvial plain, which was attained in the last few thousand years as indicated by borehole data and estimations of sedimentation rates.

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

    PubMed

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

    2012-02-01

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

  10. Biophysics, pathophysiology, and pharmacology of ion channel gating pores

    PubMed Central

    Moreau, Adrien; Gosselin-Badaroudine, Pascal; Chahine, Mohamed

    2014-01-01

    Voltage sensor domains (VSDs) are a feature of voltage gated ion channels (VGICs) and voltage sensitive proteins. They are composed of four transmembrane (TM) segments (S1–S4). Currents leaking through VSDs are called omega or gating pore currents. Gating pores are caused by mutations of the highly conserved positively charged amino acids in the S4 segment that disrupt interactions between the S4 segment and the gating charge transfer center (GCTC). The GCTC separates the intracellular and extracellular water crevices. The disruption of S4–GCTC interactions allows these crevices to communicate and create a fast activating and non-inactivating alternative cation-selective permeation pathway of low conductance, or a gating pore. Gating pore currents have recently been shown to cause periodic paralysis phenotypes. There is also increasing evidence that gating pores are linked to several other familial diseases. For example, gating pores in Nav1.5 and Kv7.2 channels may underlie mixed arrhythmias associated with dilated cardiomyopathy (DCM) phenotypes and peripheral nerve hyperexcitability (PNH), respectively. There is little evidence for the existence of gating pore blockers. Moreover, it is known that a number of toxins bind to the VSD of a specific domain of Na+ channels. These toxins may thus modulate gating pore currents. This focus on the VSD motif opens up a new area of research centered on developing molecules to treat a number of cell excitability disorders such as epilepsy, cardiac arrhythmias, and pain. The purpose of the present review is to summarize existing knowledge of the pathophysiology, biophysics, and pharmacology of gating pore currents and to serve as a guide for future studies aimed at improving our understanding of gating pores and their pathophysiological roles. PMID:24772081

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

    PubMed

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

    2016-09-01

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

  13. 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.

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

    PubMed

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

    2010-09-01

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

  15. Gas Hydrate and Pore Pressure

    NASA Astrophysics Data System (ADS)

    Tinivella, Umberta; Giustiniani, Michela

    2014-05-01

    Many efforts have been devoted to quantify excess pore pressures related to gas hydrate dissociation in marine sediments below the BSR using several approaches. Dissociation of gas hydrates in proximity of the BSR, in response to a change in the physical environment (i.e., temperature and/or pressure regime), can liberate excess gas incrising the local pore fluid pressure in the sediment, so decreasing the effective normal stress. So, gas hydrate dissociation may lead to excess pore pressure resulting in sediment deformation or failure, such as submarine landslides, sediment slumping, pockmarks and mud volcanoes, soft-sediment deformation and giant hummocks. Moreover, excess pore pressure may be the result of gas hydrate dissociation due to continuous sedimentation, tectonic uplift, sea level fall, heating or inhibitor injection. In order to detect the presence of the overpressure below the BSR, we propose two approachs. The fist approach models the BSR depth versus pore pressure; in fact, if the free gas below the BSR is in overpressure condition, the base of the gas hydrate stability is deeper with respect to the hydrostatic case. This effect causes a discrepancy between seismic and theoretical BSR depths. The second approach models the velocities versus gas hydrate and free gas concentrations and pore pressure, considering the approximation of the Biot theory in case of low frequency, i.e. seismic frequency. Knowing the P and S seismic velocity from seismic data analysis, it is possibile to jointly estimate the gas hydrate and free gas concentrations and the pore pressure regime. Alternatively, if the S-wave velocity is not availbale (due to lack of OBS/OBC data), an AVO analysis can be performed in order to extract information about Poisson ratio. Our modeling suggests that the areas characterized by shallow waters (i.e., areas in which human infrastructures, such as pipelines, are present) are significantly affected by the presence of overpressure condition

  16. Mutations in domain I interhelical loops affect the rate of pore formation by the Bacillus thuringiensis Cry1Aa toxin in insect midgut brush border membrane vesicles.

    PubMed

    Lebel, Geneviève; Vachon, Vincent; Préfontaine, Gabrielle; Girard, Frédéric; Masson, Luke; Juteau, Marc; Bah, Aliou; Larouche, Geneviève; Vincent, Charles; Laprade, Raynald; Schwartz, Jean-Louis

    2009-06-01

    Pore formation in the apical membrane of the midgut epithelial cells of susceptible insects constitutes a key step in the mode of action of Bacillus thuringiensis insecticidal toxins. In order to study the mechanism of toxin insertion into the membrane, at least one residue in each of the pore-forming-domain (domain I) interhelical loops of Cry1Aa was replaced individually by cysteine, an amino acid which is normally absent from the activated Cry1Aa toxin, using site-directed mutagenesis. The toxicity of most mutants to Manduca sexta neonate larvae was comparable to that of Cry1Aa. The ability of each of the activated mutant toxins to permeabilize M. sexta midgut brush border membrane vesicles was examined with an osmotic swelling assay. Following a 1-h preincubation, all mutants except the V150C mutant were able to form pores at pH 7.5, although the W182C mutant had a weaker activity than the other toxins. Increasing the pH to 10.5, a procedure which introduces a negative charge on the thiol group of the cysteine residues, caused a significant reduction in the pore-forming abilities of most mutants without affecting those of Cry1Aa or the I88C, T122C, Y153C, or S252C mutant. The rate of pore formation was significantly lower for the F50C, Q151C, Y153C, W182C, and S252C mutants than for Cry1Aa at pH 7.5. At the higher pH, all mutants formed pores significantly more slowly than Cry1Aa, except the I88C mutant, which formed pores significantly faster, and the T122C mutant. These results indicate that domain I interhelical loop residues play an important role in the conformational changes leading to toxin insertion and pore formation.

  17. Primary structure analysis of an integral membrane glycoprotein of the nuclear pore

    PubMed Central

    1989-01-01

    The complete primary structure of an integral membrane glycoprotein of the nuclear pore was deduced from the cDNA sequence. The cDNA encodes a polypeptide of 204,205 D containing a 25-residue-long signal sequence, two hydrophobic segments that could function as transmembrane segments, and 13 potential N-linked oligosaccharide addition sites. Endoglycosidase H reduces the molecular mass by approximately 9 kD suggesting that not all of these 13 sites are used. We discuss possible models for the topology of this protein in the pore membrane as well as a possible role in the formation of pores and pore complexes. PMID:2738089

  18. 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.

  19. Hydrochromic conjugated polymers for human sweat pore mapping.

    PubMed

    Lee, Joosub; Pyo, Minkyeong; Lee, Sang-hwa; Kim, Jaeyong; Ra, Moonsoo; Kim, Whoi-Yul; Park, Bum Jun; Lee, Chan Woo; Kim, Jong-Man

    2014-04-29

    Hydrochromic materials have been actively investigated in the context of humidity sensing and measuring water contents in organic solvents. Here we report a sensor system that undergoes a brilliant blue-to-red colour transition as well as 'Turn-On' fluorescence upon exposure to water. Introduction of a hygroscopic element into a supramolecularly assembled polydiacetylene results in a hydrochromic conjugated polymer that is rapidly responsive (<20 μs), spin-coatable and inkjet-compatible. Importantly, the hydrochromic sensor is found to be suitable for mapping human sweat pores. The exceedingly small quantities (sub-nanolitre) of water secreted from sweat pores are sufficient to promote an instantaneous colorimetric transition of the polymer. As a result, the sensor can be used to construct a precise map of active sweat pores on fingertips. The sensor technology, developed in this study, has the potential of serving as new method for fingerprint analysis and for the clinical diagnosis of malfunctioning sweat pores.

  20. Hydrochromic conjugated polymers for human sweat pore mapping

    NASA Astrophysics Data System (ADS)

    Lee, Joosub; Pyo, Minkyeong; Lee, Sang-Hwa; Kim, Jaeyong; Ra, Moonsoo; Kim, Whoi-Yul; Park, Bum Jun; Lee, Chan Woo; Kim, Jong-Man

    2014-04-01

    Hydrochromic materials have been actively investigated in the context of humidity sensing and measuring water contents in organic solvents. Here we report a sensor system that undergoes a brilliant blue-to-red colour transition as well as ‘Turn-On’ fluorescence upon exposure to water. Introduction of a hygroscopic element into a supramolecularly assembled polydiacetylene results in a hydrochromic conjugated polymer that is rapidly responsive (<20 μs), spin-coatable and inkjet-compatible. Importantly, the hydrochromic sensor is found to be suitable for mapping human sweat pores. The exceedingly small quantities (sub-nanolitre) of water secreted from sweat pores are sufficient to promote an instantaneous colorimetric transition of the polymer. As a result, the sensor can be used to construct a precise map of active sweat pores on fingertips. The sensor technology, developed in this study, has the potential of serving as new method for fingerprint analysis and for the clinical diagnosis of malfunctioning sweat pores.

  1. Assessing sulfate reduction and methane cycling in a high salinity pore water system in the northern Gulf of Mexico

    USGS Publications Warehouse

    Pohlman, J.W.; Ruppel, C.; Hutchinson, D.R.; Downer, R.; Coffin, R.B.

    2008-01-01

    Pore waters extracted from 18 piston cores obtained on and near a salt-cored bathymetric high in Keathley Canyon lease block 151 in the northern Gulf of Mexico contain elevated concentrations of chloride (up to 838 mM) and have pore water chemical concentration profiles that exhibit extensive departures (concavity) from steady-state (linear) diffusive equilibrium with depth. Minimum ??13C dissolved inorganic carbon (DIC) values of -55.9??? to -64.8??? at the sulfate-methane transition (SMT) strongly suggest active anaerobic oxidation of methane (AOM) throughout the study region. However, the nonlinear pore water chemistry-depth profiles make it impossible to determine the vertical extent of active AOM or the potential role of alternate sulfate reduction pathways. Here we utilize the conservative (non-reactive) nature of dissolved chloride to differentiate the effects of biogeochemical activity (e.g., AOM and/or organoclastic sulfate reduction) relative to physical mixing in high salinity Keathley Canyon sediments. In most cases, the DIC and sulfate concentrations in pore waters are consistent with a conservative mixing model that uses chloride concentrations at the seafloor and the SMT as endmembers. Conservative mixing of pore water constituents implies that an undetermined physical process is primarily responsible for the nonlinearity of the pore water-depth profiles. In limited cases where the sulfate and DIC concentrations deviated from conservative mixing between the seafloor and SMT, the ??13C-DIC mixing diagrams suggest that the excess DIC is produced from a 13C-depleted source that could only be accounted for by microbial methane, the dominant form of methane identified during this study. We conclude that AOM is the most prevalent sink for sulfate and that it occurs primarily at the SMT at this Keathley Canyon site.

  2. Controlling Pore Geometries and Interpore Distances of Anodic Aluminum Oxide Templates via Three-Step Anodization.

    PubMed

    Lim, Jin-Hee; Wiley, John B

    2015-01-01

    Porous alumina membranes have attracted much attention because they are very useful templates for the fabrication of various nanostructures important to nanotechnology. However, there are challenges in controlling pore geometries and interpore distances in alumina templates while maintaining highly ordered hexagonal pore structures. Herein, a three-step anodization method is utilized to prepare anodic alumina templates with various pore morphologies (e.g., arched-shape, tree-like, branched-shape) and tunable interpore distances. Such structures are not found within the more traditional alumina templates fabricated by a two-step anodization of aluminum films. The range of interpore distances and pore diameters within the modified templates increases with increasing voltages. In contrast, under decreasing voltages, hexagonally ordered pores can also branch into several pores with smaller sizes and reduced interpore distances. Electrochemical growth of metal nanowires in the modified templates helps to highlight details of the pore structures and which pore channels are active.

  3. Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site*

    PubMed Central

    Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

    2014-01-01

    Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser105 residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T5015, the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability. PMID:24448805

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

    PubMed

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

    2016-01-01

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

  5. 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

  6. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... (20″×14″) upright format signs specified in 29 CFR 1910.145(d)(4) and this paragraph; and (iii... 40 Protection of Environment 8 2011-07-01 2011-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an...

  7. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (20″×14″) upright format signs specified in 29 CFR 1910.145(d)(4) and this paragraph; and (iii... 40 Protection of Environment 8 2010-07-01 2010-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an...

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

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... (20″×14″) upright format signs specified in 29 CFR 1910.145(d)(4) and this paragraph; and (iii... 40 Protection of Environment 9 2013-07-01 2013-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an...

  9. 77 FR 39508 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-03

    ... specific project proposals on those leases) in an identified Wind Energy Area (WEA) on the OCS offshore... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on the... Activities on the Atlantic OCS Offshore RI and MA'' to: Program Manager, Office of Renewable Energy...

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

    SciTech Connect

    Ashby, J.; Rowe, J.

    1980-02-01

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

  11. Computational approaches to the determination of active site structures and reaction mechanisms in heterogeneous catalysts.

    PubMed

    Catlow, C R A; French, S A; Sokol, A A; Thomas, J M

    2005-04-15

    We apply quantum chemical methods to the study of active site structures and reaction mechanisms in mesoporous silica and metal oxide catalysts. Our approach is based on the use of both molecular cluster and embedded cluster (QM/MM) techniques, where the active site and molecular complex are described using density functional theory (DFT) and the embedding matrix simulated by shell model potentials. We consider three case studies: alkene epoxidation over the microporous TS-1 catalyst; methanol synthesis on ZnO and Cu/ZnO and C-H bond activation over Li-doped MgO.

  12. Computational approaches to the determination of active site structures and reaction mechanisms in heterogeneous catalysts.

    PubMed

    Catlow, C R A; French, S A; Sokol, A A; Thomas, J M

    2005-04-15

    We apply quantum chemical methods to the study of active site structures and reaction mechanisms in mesoporous silica and metal oxide catalysts. Our approach is based on the use of both molecular cluster and embedded cluster (QM/MM) techniques, where the active site and molecular complex are described using density functional theory (DFT) and the embedding matrix simulated by shell model potentials. We consider three case studies: alkene epoxidation over the microporous TS-1 catalyst; methanol synthesis on ZnO and Cu/ZnO and C-H bond activation over Li-doped MgO. PMID:15901543

  13. Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge.

    PubMed

    Tara, S; Elcock, A H; Kirchhoff, P D; Briggs, J M; Radic, Z; Taylor, P; McCammon, J A

    1998-12-01

    It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant kon, for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction.

  14. 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.

  15. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-16

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

  18. The Type III Secretion Translocation Pore Senses Host Cell Contact

    PubMed Central

    Armentrout, Erin I.; Rietsch, Arne

    2016-01-01

    Type III secretion systems (T3SS) are nano-syringes used by a wide range of Gram-negative pathogens to promote infection by directly injecting effector proteins into targeted host cells. Translocation of effectors is triggered by host-cell contact and requires assembly of a pore in the host-cell plasma membrane, which consists of two translocator proteins. Our understanding of the translocation pore, how it is assembled in the host cell membrane and its precise role in effector translocation, is extremely limited. Here we use a genetic technique to identify protein-protein contacts between pore-forming translocator proteins, as well as the T3SS needle-tip, that are critical for translocon function. The data help establish the orientation of the translocator proteins in the host cell membrane. Analysis of translocon function in mutants that break these contacts demonstrates that an interaction between the pore-forming translocator PopD and the needle-tip is required for sensing host cell contact. Moreover, tethering PopD at a dimer interface also specifically prevents host-cell sensing, arguing that the translocation pore is actively involved in detecting host cell contact. The work presented here therefore establishes a signal transduction pathway for sensing host cell contact that is initiated by a conformational change in the translocation pore, and is subsequently transmitted to the base of the apparatus via a specific contact between the pore and the T3SS needle-tip. PMID:27022930

  19. 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.

  20. 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.

  1. Modeling permeability evolution by precipitation and dissolution: from a single-pore channel to heterogeneous multi-pore systems

    NASA Astrophysics Data System (ADS)

    Silin, D.; Molins, S.

    2011-12-01

    Permeability modification by mineral precipitation or dissolution can significantly affect injection well performance in a bioremediation project. Well clogging may occur due to massive mineral deposition on the pore walls near the wellbore. Although the size of a cleanup site can be measured in hundreds of meters or even kilometers, the underlying geochemical and hydrologic processes in individual pores eventually define where and how fast the permeability will decline. Therefore, it is important to understand these pore-scale mechanisms in order to reduce the hindering effect of pore clogging and develop efficient well treatment procedures when its injectivity drops below the minimal-tolerance level. Additionally, model-based permeability-porosity correlations for pore space evolution caused by precipitation or dissolution are needed for realistic reservoir-scale numerical simulations. To gain insights into the pore-scale mechanisms of permeability modification by mineral precipitation and dissolution, we have developed a model describing the dynamic coupling between flow and reactive transport. The model relies on a sequential approach where alternating finite-volume multicomponent reactive transport and flow simulations evaluate the local precipitation-dissolution rates, update the pore space geometry from the mass balance considerations, the re-compute the flow field on the updated sample model. Segmented three dimensional micro-tomography images of the sediment, the chemical composition of the fluid, and the mineral composition of the rock are the input data for simulations. Some model details were reported at previous AGU meetings. We validate the model against a single-pore calcite dissolution experiment. Building on this problem, we use the model to evaluate the precipitation-dissolution patterns for a range of flow rates. We then simulate multi-pore systems to qualitatively reproduce the pattern of precipitation observed in experiments. For that purpose

  2. Evaluation of physical activity web sites for use of behavior change theories.

    PubMed

    Doshi, Amol; Patrick, Kevin; Sallis, James F; Calfas, Karen

    2003-01-01

    Physical activity (PA) Web sites were assessed for their use of behavior change theories, including constructs of the health belief model, Transtheoretical Model, social cognitive theory, and the theory of reasoned action and planned behavior. An evaluation template for assessing PA Web sites was developed, and content validity and interrater reliability were demonstrated. Two independent raters evaluated 24 PA Web sites. Web sites varied widely in application of theory-based constructs, ranging from 5 to 48 on a 100-point scale. The most common intervention strategies were general information, social support, and realistic goal areas. Coverage of theory-based strategies was low, varying from 26% for social cognitive theory to 39% for health belief model. Overall, PA Web sites provided little assessment, feedback, or individually tailored assistance for users. They were unable to substantially tailor the on-line experience for users at different stages of change or different demographic characteristics.

  3. Catalytic performance of Metal-Organic-Frameworks vs. extra-large pore zeolite UTL in condensation reactions.

    PubMed

    Shamzhy, Mariya; Opanasenko, Maksym; Shvets, Oleksiy; Cejka, Jiří

    2013-01-01

    Catalytic behavior of isomorphously substituted B-, Al-, Ga-, and Fe-containing extra-large pore UTL zeolites was investigated in Knoevenagel condensation involving aldehydes, Pechmann condensation of 1-naphthol with ethylacetoacetate, and Prins reaction of β-pinene with formaldehyde and compared with large-pore aluminosilicate zeolite beta and representative Metal-Organic-Frameworks Cu3(BTC)2 and Fe(BTC). The yield of the target product over the investigated catalysts in Knoevenagel condensation increases in the following sequence: (Al)beta < (Al)UTL < (Ga)UTL < (Fe)UTL < Fe(BTC) < (B)UTL < Cu3(BTC)2 being mainly related to the improving selectivity with decreasing strength of active sites of the individual catalysts. The catalytic performance of Fe(BTC), containing the highest concentration of Lewis acid sites of the appropriate strength is superior over large-pore zeolite (Al)beta and B-, Al-, Ga-, Fe-substituted extra-large pore zeolites UTL in Prins reaction of β-pinene with formaldehyde and Pechmann condensation of 1-naphthol with ethylacetoacetate.

  4. Catalytic performance of Metal-Organic-Frameworks vs. extra-large pore zeolite UTL in condensation reactions

    PubMed Central

    Shamzhy, Mariya; Opanasenko, Maksym; Shvets, Oleksiy; Čejka, Jiří

    2013-01-01

    Catalytic behavior of isomorphously substituted B-, Al-, Ga-, and Fe-containing extra-large pore UTL zeolites was investigated in Knoevenagel condensation involving aldehydes, Pechmann condensation of 1-naphthol with ethylacetoacetate, and Prins reaction of β-pinene with formaldehyde and compared with large-pore aluminosilicate zeolite beta and representative Metal-Organic-Frameworks Cu3(BTC)2 and Fe(BTC). The yield of the target product over the investigated catalysts in Knoevenagel condensation increases in the following sequence: (Al)beta < (Al)UTL < (Ga)UTL < (Fe)UTL < Fe(BTC) < (B)UTL < Cu3(BTC)2 being mainly related to the improving selectivity with decreasing strength of active sites of the individual catalysts. The catalytic performance of Fe(BTC), containing the highest concentration of Lewis acid sites of the appropriate strength is superior over large-pore zeolite (Al)beta and B-, Al-, Ga-, Fe-substituted extra-large pore zeolites UTL in Prins reaction of β-pinene with formaldehyde and Pechmann condensation of 1-naphthol with ethylacetoacetate. PMID:24790940

  5. Catalytic performance of Metal-Organic-Frameworks vs. extra-large pore zeolite UTL in condensation reactions

    NASA Astrophysics Data System (ADS)

    Shamzhy, Mariya; Opanasenko, Maksym; Shvets, Oleksiy; Čejka, Jiří

    2013-08-01

    Catalytic behavior of isomorphously substituted B-, Al-, Ga-, and Fe-containing extra-large pore UTL zeolites was investigated in Knoevenagel condensation involving aldehydes, Pechmann condensation of 1-naphthol with ethylacetoacetate, and Prins reaction of β-pinene with formaldehyde and compared with large-pore aluminosilicate zeolite BEA and representative Metal-Organic-Frameworks Cu3(BTC)2 and Fe(BTC). The yield of the target product over the investigated catalysts in Knoevenagel condensation increases in the following sequence: (Al)BEA < (Al)UTL < (Ga)UTL < (Fe)UTL < Fe(BTC) < (B)UTL < Cu3(BTC)2 being mainly related to the improving selectivity with decreasing strength of active sites of the individual catalysts. The catalytic performance of Fe(BTC), containing the highest concentration of Lewis acid sites of the appropriate strength is superior over large-pore zeolite (Al)BEA and B-, Al-, Ga-, Fe-substituted extra-large pore zeolites UTL in Prins reaction of β-pinene with formaldehyde and Pechmann condensation of 1-naphthol with ethylacetoacetate.

  6. Site- and kinase-specific phosphorylation-mediated activation of SLAC1, a guard cell anion channel stimulated by abscisic acid.

    PubMed

    Maierhofer, Tobias; Diekmann, Marion; Offenborn, Jan Niklas; Lind, Christof; Bauer, Hubert; Hashimoto, Kenji; S Al-Rasheid, Khaled A; Luan, Sheng; Kudla, Jörg; Geiger, Dietmar; Hedrich, Rainer

    2014-09-09

    Under drought stress, abscisic acid (ABA) triggers closure of leaf cell pores called stomata, which are formed by two specialized cells called guard cells in plant epidermis. Two pathways downstream of ABA stimulate phosphorylation of the S-type anion channels SLAC1 (slow anion channel associated 1) and SLAH3 (SLAC1 homolog 3), which causes these channels to open, reducing guard cell volume and triggering stomatal closure. One branch involves OST1 (open stomata 1), a calcium-independent SnRK2-type kinase, and the other branch involves calcium-dependent protein kinases of the CPK (calcium-dependent protein kinase) family. We used coexpression analyses in Xenopus oocytes to show that the calcineurin B-like (CBL) calcium sensors CBL1 and CBL9 and their interacting protein kinase CIPK23 also triggered SLAC1 and SLAH3 opening. We analyzed whether regulation of SLAC1 opening by these different families of kinases involved the same or different sites on SLAC1 by measuring channel conductance of SLAC1 with mutations in the putative phosphorylation sites in the amino or carboxyl termini coexpressed with specific kinases in Xenopus oocytes. SLAC1 mutants lacking the OST1-phosphorylated site were still activated by CPK or by CBL/CIPK complexes. Phosphorylation and activation of SLAC1 by any of the kinases were inhibited by the phosphatase ABI1 (ABA insensitive 1), which is inactivated in response to ABA signaling. These findings identified CBL/CIPK complexes as potential regulators of stomatal aperture through S-type anion channels and indicated that phosphorylation at distinct sites enables SLAC1 activation by both calcium-dependent and calcium-independent pathways downstream of ABA.

  7. Membrane pores induced by magainin

    SciTech Connect

    Ludtke, S.J.; He, Ke; Heller, W.T.

    1996-10-29

    Magainin, found in the skin of Xenopus laevis, belongs to a broad class of antimicrobial peptides which kill bacteria by permeabilizing the cytoplasmic membrane but do not lyse eukaryotic cells. The 23-residue peptide has been shown to form an amphiphilic helix when associated with membranes. However, its molecular mechanism of action has been controversial. Oriented circular dichroism has detected helical magainin oriented perpendicular to the plane of the membrane at high peptide concentrations, but Raman, fluorescence, differential scanning calorimetry, and NMR all indicate that the peptide is associated with the head groups of the lipid bilayer. Here we show that neutron in-plane scattering detects pores formed by magainin 2 in membranes only when a substantial fraction of the peptide is oriented perpendicular to the membrane. The pores are almost twice as large as the alamethicin pores. On the basis of the in-plane scattering data, we propose a toroidal (or wormhole) model, which differs from the barrel-stave model of alamethicin in that the lipid bends back on itself like the inside of a torus. The bending requires a lateral expansion in the head group region of the bilayer. Magainin monomers play the role of fillers in the expansion region thereby stabilizing the pore. This molecular configuration is consistent with all published magainin data. 33 refs., 5 figs.

  8. 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.

  9. A Pore-Network Model of In-Situ Combustion in Porous Media

    SciTech Connect

    Lu, Chuan; Yortsos, Y.C.

    2001-01-29

    This report the use of dual pore networks (pores and solid sites) for modeling the effect of the microstructure on combustion processes in porous media is considered. The model accounts for flow and transport of the gas phase in the porespace, where convection predominates, and for heat transfer by conduction in the solid phase. Gas phase flow in the pore and throats is governed by Darcy's law.

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

    SciTech Connect

    Eaton, Todd R.; Boston, Andrew M.; Thompson, Anthony B.; Gray, Kimberly A.; Notestein, Justin M.

    2015-06-04

    Quantifying specific active sites in supported catalysts improves our understanding and assists in rational design. Supported oxides can undergo significant structural changes as surface densities increase from site-isolated cations to monolayers and crystallites, which changes the number of kinetically relevant sites. Herein, TiOx domains are titrated on TiOx–SiO2 selectively with phenylphosphonic acid (PPA). An ex situ method quantifies all fluid-accessible TiOx, whereas an in situ titration during cis-cyclooctene epoxidation provides previously unavailable values for the number of tetrahedral Ti sites on which H2O2 activation occurs. We use this method to determine the active site densities of 22 different catalysts with different synthesis methods, loadings, and characteristic spectra and find a single intrinsic turnover frequency for cis-cyclooctene epoxidation of (40±7) h-1. This simple method gives molecular-level insight into catalyst structure that is otherwise hidden when bulk techniques are used.

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

    PubMed Central

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

    1987-01-01

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

  12. 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

  13. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase.

    PubMed

    Fenwick, Michael K; Mehta, Angad P; Zhang, Yang; Abdelwahed, Sameh H; Begley, Tadhg P; Ealick, Steven E

    2015-03-27

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

  14. Solvent Tuning of Electrochemical Potentials in the Active Sites of HiPIP Versus Ferredoxin

    SciTech Connect

    Dey, A.; Francis, E.J.; Adams, M.W.W.; Babini, E.; Takahashi, Y.; Fukuyama, K.; Hodgson, K.O.; Hedman, B.; Solomon, E.I.; /Stanford U., Chem. Dept. /Georgia U. /Bologna U. /Osaka U. /SLAC, SSRL

    2009-04-29

    A persistent puzzle in the field of biological electron transfer is the conserved iron-sulfur cluster motif in both high potential iron-sulfur protein (HiPIP) and ferredoxin (Fd) active sites. Despite this structural similarity, HiPIPs react oxidatively at physiological potentials, whereas Fds are reduced. Sulfur K-edge x-ray absorption spectroscopy uncovers the substantial influence of hydration on this variation in reactivity. Fe-S covalency is much lower in natively hydrated Fd active sites than in HiPIPs but increases upon water removal; similarly, HiPIP covalency decreases when unfolding exposes an otherwise hydrophobically shielded active site to water. Studies on model compounds and accompanying density functional theory calculations support a correlation of Fe-S covalency with ease of oxidation and therefore suggest that hydration accounts for most of the difference between Fd and HiPIP reduction potentials.

  15. 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.

  16. Wasp recruitment to the T cell:APC contact site occurs independently of Cdc42 activation.

    PubMed

    Cannon, J L; Labno, C M; Bosco, G; Seth, A; McGavin, M H; Siminovitch, K A; Rosen, M K; Burkhardt, J K

    2001-08-01

    Cdc42 and WASP are critical regulators of actin polymerization whose function during T cell signaling is poorly understood. Using a novel reagent that specifically detects Cdc42-GTP in fixed cells, we found that activated Cdc42 localizes to the T cell:APC contact site in an antigen-dependent manner. TCR signaling alone was sufficient to induce localization of Cdc42-GTP, and functional Lck and Zap-70 kinases were required. WASP also localized to the T cell:APC contact site in an antigen-dependent manner. Surprisingly, WASP localization was independent of the Cdc42 binding domain but required the proline-rich domain. Our results indicate that localized WASP activation requires the integration of multiple signals: WASP is recruited via interaction with SH3 domain-containing proteins and is activated by Cdc42-GTP concentrated at the same site. PMID:11520460

  17. Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

    PubMed

    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poddar, Harshwardhan; Baas, Bert-Jan; Poelarends, Gerrit J

    2016-07-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site. PMID:27238293

  18. Pro-apoptotic Bax molecules densely populate the edges of membrane pores

    PubMed Central

    Kuwana, Tomomi; Olson, Norman H.; Kiosses, William B.; Peters, Bjoern; Newmeyer, Donald D.

    2016-01-01

    How the pro-apoptotic Bax protein permeabilizes the mitochondrial outer membrane is not fully understood. Previously, using cryo-electron microscopy (cryo-EM), we showed that activated Bax forms large, growing pores. Whether formed in liposomes or in mitochondrial outer membranes, Bax-induced pores exhibit the same morphology, with negative curvature flanking the edges and with no visible protein structure protruding from the membranes. Here we used cryo-EM to show that gold-labeled Bax molecules, after activation by Bid, became localized strictly at pore edges. This argues that Bax acts at short range to deform the membrane. Also, Bax molecules populated the walls of both small and large pores at the same density, implying that Bax is continuously recruited to the pores as they widen. Moreover, because all Bax molecules became oligomerized after membrane insertion, we infer that Bax oligomers are present at pore edges. We suggest that oligomerization may promote pore enlargement. PMID:27255832

  19. 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

  20. 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.

  1. The active site of low-temperature methane hydroxylation in iron-containing zeolites.

    PubMed

    Snyder, Benjamin E R; Vanelderen, Pieter; Bols, Max L; Hallaert, Simon D; Böttger, Lars H; Ungur, Liviu; Pierloot, Kristine; Schoonheydt, Robert A; Sels, Bert F; Solomon, Edward I

    2016-08-18

    An efficient catalytic process for converting methane into methanol could have far-reaching economic implications. Iron-containing zeolites (microporous aluminosilicate minerals) are noteworthy in this regard, having an outstanding ability to hydroxylate methane rapidly at room temperature to form methanol. Reactivity occurs at an extra-lattice active site called α-Fe(ii), which is activated by nitrous oxide to form the reactive intermediate α-O; however, despite nearly three decades of research, the nature of the active site and the factors determining its exceptional reactivity are unclear. The main difficulty is that the reactive species-α-Fe(ii) and α-O-are challenging to probe spectroscopically: data from bulk techniques such as X-ray absorption spectroscopy and magnetic susceptibility are complicated by contributions from inactive 'spectator' iron. Here we show that a site-selective spectroscopic method regularly used in bioinorganic chemistry can overcome this problem. Magnetic circular dichroism reveals α-Fe(ii) to be a mononuclear, high-spin, square planar Fe(ii) site, while the reactive intermediate, α-O, is a mononuclear, high-spin Fe(iv)=O species, whose exceptional reactivity derives from a constrained coordination geometry enforced by the zeolite lattice. These findings illustrate the value of our approach to exploring active sites in heterogeneous systems. The results also suggest that using matrix constraints to activate metal sites for function-producing what is known in the context of metalloenzymes as an 'entatic' state-might be a useful way to tune the activity of heterogeneous catalysts. PMID:27535535

  2. 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.

  3. A Tale of Two Isomerases: Compact versus Extended Active Sites in Ketosteroid Isomerase and Phosphoglucose Isomerase

    SciTech Connect

    Somarowthu, Srinivas; Brodkin, Heather R.; D’Aquino, J. Alejandro; Ringe, Dagmar; Ondrechen, Mary Jo; Beuning, Penny J.

    2012-07-11

    Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

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

    PubMed Central

    Nosrati, Geoffrey R; Houk, K N

    2012-01-01

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

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

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-05-01

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

  6. Preliminary examination of the impacts of repository site characterization activities and facility construction and operation activities on Hanford air quality

    SciTech Connect

    Glantz, C.S.; Ramsdell, J.V.

    1986-04-01

    Air quality impacts that would result from site characterization activities and from the construction and operation of a high-level nuclear wste repository at Hanford are estimated using two simple atmospheric dispersion models, HANCHI and CHISHORT. Model results indicate that pollutant concentrations would not exceed ambient air quality standards at any point outside the Hanford fenceline or at any publicly accessible location within the Hanford Site. The increase in pollutant concentrations in nearby communities due to site activities would be minimal. HANCHI and CHISHORT are documented in the appendices of this document. Further study of the repository's impact on air quality will be conducted when more detailed project plans and work schedules are available.

  7. Applications of biological pores in nanomedicine, sensing, and nanoelectronics

    PubMed Central

    Majd, Sheereen; Yusko, Erik C; Billeh, Yazan N; Macrae, Michael X; Yang, Jerry; Mayer, Michael

    2011-01-01

    Biological protein pores and pore-forming peptides can generate a pathway for the flux of ions and other charged or polar molecules across cellular membranes. In nature, these nanopores have diverse and essential functions that range from maintaining cell homeostasis and participating in cell signaling to activating or killing cells. The combination of the nanoscale dimensions and sophisticated – often regulated – functionality of these biological pores make them particularly attractive for the growing field of nanobiotechnology. Applications range from single-molecule sensing to drug delivery and targeted killing of malignant cells. Potential future applications may include the use of nanopores for single strand DNA sequencing and for generating bio-inspired, and possibly, biocompatible visual detection systems and batteries. This article reviews the current state of applications of pore-forming peptides and proteins in nanomedicine, sensing, and nanoelectronics. PMID:20561776

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

    PubMed

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

    2015-12-15

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

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

    PubMed Central

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

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr890, Ser893, Thr894 and Thr899, differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  10. Displacement of soil pore water by trichloroethylene

    USGS Publications Warehouse

    Wershaw, R. L.; Aiken, G.R.; Imbrigiotta, T.E.; Goldberg, M.C.

    1994-01-01

    Dense nonaqueous phase liquids (DNAPLS) are important pollutants because of their widespread use as chemical and industrial solvents. An example of the pollution caused by the discharge of DNAPLs is found at the Picatinny Arsenal, New Jersey, where trichloroethylene (TCE) has been discharged directly into the unsaturated zone. This discharge has resulted in the formation of a plume of TCE-contaminated water in the aquifer downgradient of the discharge. A zone of dark-colored groundwater containing a high dissolved organic C content has been found near the point of discharge of the TCE. The colored-water plume extends from the point of discharge at least 30 m (100 feet) downgradient. Fulvic acids isolated from the colored-waters plume, from water from a background well that has not been affected by the discharge of chlorinated solvents, and from soil pore water collected in a lysimeter installed at an uncontaminated site upgradient of the study area have been compared. Nuclear magnetic resonance spectra of the fulvic acids from the colored waters and from the lysimeter are very similar, but are markedly different from the nuclear magnetic resonance spectrum of the fulvic acid from the background well. The three-dimensional fluorescence spectrum and the DOC fractionation profile of the colored groundwater and the soil pore water are very similar to each other, but quite different from those of the background water. It is proposed from these observations that this colored water is soil pore water that has been displaced by a separate DNAPL liquid phase downward to the saturated zone.

  11. Ion binding in the Open HCN Pacemaker Channel Pore: Fast Mechanisms to Shape “Slow” Channels

    PubMed Central

    Lyashchenko, Alex K.; Tibbs, Gareth R.

    2008-01-01

    IH pacemaker channels carry a mixed monovalent cation current that, under physiological ion gradients, reverses at ∼−34 mV, reflecting a 4:1 selectivity for K over Na. However, IH channels display anomalous behavior with respect to permeant ions such that (a) open channels do not exhibit the outward rectification anticipated assuming independence; (b) gating and selectivity are sensitive to the identity and concentrations of externally presented permeant ions; (c) the channels' ability to carry an inward Na current requires the presence of external K even though K is a minor charge carrier at negative voltages. Here we show that open HCN channels (the hyperpolarization-activated, cyclic nucleotide sensitive pore forming subunits of IH) undergo a fast, voltage-dependent block by intracellular Mg in a manner that suggests the ion binds close to, or within, the selectivity filter. Eliminating internal divalent ion block reveals that (a) the K dependence of conduction is mediated via K occupancy of site(s) within the pore and that asymmetrical occupancy and/or coupling of these sites to flux further shapes ion flow, and (b) the kinetics of equilibration between K-vacant and K-occupied states of the pore (10–20 μs or faster) is close to the ion transit time when the pore is occupied by K alone (∼0.5–3 μs), a finding that indicates that either ion:ion repulsion involving Na is adequate to support flux (albeit at a rate below our detection threshold) and/or the pore undergoes rapid, permeant ion-sensitive equilibration between nonconducting and conducting configurations. Biophysically, further exploration of the Mg site and of interactions of Na and K within the pore will tell us much about the architecture and operation of this unusual pore. Physiologically, these results suggest ways in which “slow” pacemaker channels may contribute dynamically to the shaping of fast processes such as Na-K or Ca action potentials. PMID:18270171

  12. 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

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

    PubMed Central

    Colombo, Matteo; Girard, Eric; Franzetti, Bruno

    2016-01-01

    TET aminopeptidases are dodecameric particles shared in the three life domains involved in various biological processes, from carbon source provider in archaea to eye-pressure regulation in humans. Each subunit contains a dinuclear metal site (M1 and M2) responsible for the enzyme catalytic activity. However, the role of each metal ion is still uncharacterized. Noteworthy, while mesophilic TETs are activated by Mn2+, hyperthermophilic TETs prefers Co2+. Here, by means of anomalous x-ray crystallography and enzyme kinetics measurements of the TET3 aminopeptidase from the hyperthermophilic organism Pyrococcus furiosus (PfTET3), we show that M2 hosts the catalytic activity of the enzyme, while M1 stabilizes the TET3 quaternary structure and controls the active site flexibility in a temperature dependent manner. A new third metal site (M3) was found in the substrate binding pocket, modulating the PfTET3 substrate preferences. These data show that TET activity is tuned by the molecular interplay among three metal sites. PMID:26853450

  14. Human Activities in Natura 2000 Sites: A Highly Diversified Conservation Network

    NASA Astrophysics Data System (ADS)

    Tsiafouli, Maria A.; Apostolopoulou, Evangelia; Mazaris, Antonios D.; Kallimanis, Athanasios S.; Drakou, Evangelia G.; Pantis, John D.

    2013-05-01

    The Natura 2000 network was established across the European Union's (EU) Member States with the aim to conserve biodiversity, while ensuring the sustainability of human activities. However, to what kind and to what extent Natura 2000 sites are subject to human activities and how this varies across Member States remains unspecified. Here, we analyzed 111,269 human activity records from 14,727 protected sites in 20 Member States. The frequency of occurrence of activities differs among countries, with more than 86 % of all sites being subjected to agriculture or forestry. Activities like hunting, fishing, urbanization, transportation, and tourism are more frequently recorded in south European sites than in northern or eastern ones. The observed variations indicate that Natura 2000 networks are highly heterogeneous among EU Member States. Our analysis highlights the importance of agriculture in European landscapes and indicates possible targets for policy interventions at national, European, or "sub-European" level. The strong human presence in the Natura 2000 network throughout Member States, shows that conservation initiatives could succeed only by combining social and ecological sustainability and by ensuring the integration of policies affecting biodiversity.

  15. Active-Site Monovalent Cations Revealed in a 1.55 Å Resolution Hammerhead Ribozyme Structure

    PubMed Central

    Anderson, Michael; Schultz, Eric P.; Martick, Monika; Scott, William G.

    2013-01-01

    We have obtained a 1.55 Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni in conditions that permit detailed observations of Na+ ion binding in the ribozyme's active site. At least two such Na+ ions are observed. The first Na+ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical to that previously observed for divalent cations. A second Na+ ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na+, but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na+ directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest resolution ribozyme structure in the protein data bank. PMID:23711504

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

    PubMed

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

    2015-02-01

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

  17. Electrohydrodynamic jet process for pore-structure-controlled 3D fibrous architecture as a tissue regenerative material: fabrication and cellular activities.

    PubMed

    Kim, Min Seong; Kim, GeunHyung

    2014-07-22

    In this study, we propose a new scaffold fabrication method, "direct electro-hydrodynamic jet process," using the initial jet of an electrospinning process and ethanol media as a target. The fabricated three-dimensional (3D) fibrous structure was configured with multilayered microsized struts consisting of randomly entangled micro/nanofibrous architecture, similar to that of native extracellular matrixes. The fabrication of the structure was highly dependent on various processing parameters, such as the surface tension of the target media, and the flow rate and weight fraction of the polymer solution. As a tissue regenerative material, the 3D fibrous scaffold was cultured with preosteoblasts to observe the initial cellular activities in comparison with a solid-freeform fabricated 3D scaffold sharing a similar structural geometry. The cell-culture results showed that the newly developed scaffold provided outstanding microcellular environmental conditions to the seeded cells (about 3.5-fold better initial cell attachment and 2.1-fold better cell proliferation).

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

    PubMed

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

    1998-12-30

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

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

    PubMed Central

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

    2003-01-01

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

  20. Immobilized low-activity waste site borehole 299-E17-21

    SciTech Connect

    Reidel, S.P.; Reynolds, K.D.; Horton, D.G.

    1998-08-01

    The Tank Waste Remediation System (TWRS) is the group at the Hanford Site responsible for the safe underground storage of liquid waste from previous Hanford Site operations, the storage and disposal of immobilized tank waste, and closure of underground tanks. The current plan is to dispose of immobilized low-activity tank waste (ILAW) in new facilities in the southcentral part of 200-East Area and in four existing vaults along the east side of 200-East Area. Boreholes 299-E17-21, B8501, and B8502 were drilled at the southwest corner of the ILAW site in support of the Performance Assessment activities for the disposal options. This report summarizes the initial geologic findings, field tests conducted on those boreholes, and ongoing studies. One deep (480 feet) borehole and two shallow (50 feet) boreholes were drilled at the southwest corner of the ILAW site. The primary factor dictating the location of the boreholes was their characterization function with respect to developing the geohydrologic model for the site and satisfying associated Data Quality Objectives. The deep borehole was drilled to characterize subsurface conditions beneath the ILAW site, and two shallow boreholes were drilled to support an ongoing environmental tracer study. The tracer study will supply information to the Performance Assessment. All the boreholes provide data on the vadose zone and saturated zone in a previously uncharacterized area.

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

    PubMed Central

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

    2006-01-01

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

  2. A facile reflux procedure to increase active surface sites form highly active and durable supported palladium@platinum bimetallic nanodendrites

    NASA Astrophysics Data System (ADS)

    Wang, Qin; Li, Yingjun; Liu, Baocang; Xu, Guangran; Zhang, Geng; Zhao, Qi; Zhang, Jun

    2015-11-01

    A series of well-dispersed bimetallic Pd@Pt nanodendrites uniformly supported on XC-72 carbon black are fabricated by using different capping agents. These capping agents are essential for the branched morphology control. However, the surfactant adsorbed on the nanodendrites surface blocks the access of reactant molecules to the active surface sites, and the catalytic activities of these bimetallic nanodendrites are significantly restricted. Herein, a facile reflux procedure to effectively remove the capping agent molecules without significantly affecting their sizes is reported for activating supported nanocatalysts. More significantly, the structure and morphology of the nanodendrites can also be retained, enhancing the numbers of active surface sites, catalytic activity and stability toward methanol and ethanol electro-oxidation reactions. The as-obtained hot water reflux-treated Pd@Pt/C catalyst manifests superior catalytic activity and stability both in terms of surface and mass specific activities, as compared to the untreated catalysts and the commercial Pt/C and Pd/C catalysts. We anticipate that this effective and facile removal method has more general applicability to highly active nanocatalysts prepared with various surfactants, and should lead to improvements in environmental protection and energy production.

  3. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions

    PubMed Central

    Herter, Susanne; Kranz, David C; Turner, Nicholas J

    2015-01-01

    Summary Cytochrome P450 monooxygenases are useful biocatalysts for C–H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations. PMID:26664590

  4. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions.

    PubMed

    Kelly, Paul P; Eichler, Anja; Herter, Susanne; Kranz, David C; Turner, Nicholas J; Flitsch, Sabine L

    2015-01-01

    Cytochrome P450 monooxygenases are useful biocatalysts for C-H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations.

  5. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions.

    PubMed

    Kelly, Paul P; Eichler, Anja; Herter, Susanne; Kranz, David C; Turner, Nicholas J; Flitsch, Sabine L

    2015-01-01

    Cytochrome P450 monooxygenases are useful biocatalysts for C-H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations. PMID:26664590

  6. Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels.

    PubMed

    Saam, Jan; Ivanov, Igor; Walther, Matthias; Holzhütter, Hermann-Georg; Kuhn, Hartmut

    2007-08-14

    Cells contain numerous enzymes that use molecular oxygen for their reactions. Often, their active sites are buried deeply inside the protein, which raises the question whether there are specific access channels guiding oxygen to the site of catalysis. Choosing 12/15-lipoxygenase as a typical example for such oxygen-dependent enzymes, we determined the oxygen distribution within the protein and defined potential routes for oxygen access. For this purpose, we have applied an integrated strategy of structural modeling, molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements. First, we computed the 3D free-energy distribution for oxygen, which led to identification of four oxygen channels in the protein. All channels connect the protein surface with a region of high oxygen affinity at the active site. This region is localized opposite to the nonheme iron providing a structural explanation for the reaction specificity of this lipoxygenase isoform. The catalytically most relevant path can be obstructed by L367F exchange, which leads to a strongly increased Michaelis constant for oxygen. The blocking mechanism is explained in detail by reordering the hydrogen-bonding network of water molecules. Our results provide strong evidence that the main route for oxygen access to the active site of the enzyme follows a channel formed by transiently interconnected cavities whereby the opening and closure are governed by side chain dynamics. PMID:17675410

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

    PubMed

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

    2016-08-24

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

  8. A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon.

    PubMed

    Rashid, N; Morikawa, M; Kanaya, S; Atomi, H; Imanaka, T

    1999-02-19

    A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein.

  9. Docking and molecular dynamics studies at trypanothione reductase and glutathione reductase active sites.

    PubMed

    Iribarne, Federico; Paulino, Margot; Aguilera, Sara; Murphy, Miguel; Tapia, Orlando

    2002-05-01

    A theoretical docking study on the active sites of trypanothione reductase (TR) and glutathione reductase (GR) with the corresponding natural substrates, trypanothione disulfide (T[S]2) and glutathione disulfide (GSSG), is reported. Molecular dynamics simulations were carried out in order to check the robustness of the docking results. The energetic results are in agreement with previous experimental findings and show the crossed complexes have lower stabilization energies than the natural ones. To test DOCK3.5, four nitro furanic compounds, previously designed as potentially active anti-chagasic molecules, were docked at the GR and TR active sites with the DOCK3.5 procedure. A good correlation was found between differential inhibitory activity and relative interaction energy (affinity). The results provide a validation test for the use of DOCK3.5 in connection with the design of anti-chagasic drugs.

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

    PubMed

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

    2015-03-23

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

  11. Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo

    PubMed Central

    Kono, Mari; Tucker, Ana E.; Tran, Jennifer; Bergner, Jennifer B.; Turner, Ewa M.; Proia, Richard L.

    2014-01-01

    Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S1P1 recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease. PMID:24667638

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

    PubMed

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

    2007-10-01

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

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

    PubMed

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

    2016-07-19

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

  14. Identification of active sites in gold-catalyzed hydrogenation of acrolein.

    PubMed

    Mohr, Christian; Hofmeister, Herbert; Radnik, Jörg; Claus, Peter

    2003-02-19

    The active sites of supported gold catalysts, favoring the adsorption of C=O groups of acrolein and subsequent reaction to allyl alcohol, have been identified as edges of gold nanoparticles. After our recent finding that this reaction preferentially occurs on single crystalline particles rather than multiply twinned ones, this paper reports on a new approach to distinguish different features of the gold particle morphology. Elucidation of the active site issue cannot be simply done by varying the size of gold particles, since the effects of faceting and multiply twinned particles may interfere. Therefore, modification of the gold particle surface by indium has been used to vary the active site characteristics of a suitable catalyst, and a selective decoration of gold particle faces has been observed, leaving edges free. This is in contradiction to theoretical predictions, suggesting a preferred occupation of the low-coordinated edges of the gold particles. On the bimetallic catalyst, the desired allyl alcohol is the main product (selectivity 63%; temperature 593 K, total pressure p(total) = 2 MPa). From the experimentally proven correlation between surface structure and catalytic behavior, the edges of single crystalline gold particles have been identified as active sites for the preferred C=O hydrogenation. PMID:12580618

  15. Evidence for surface Ag + complexes as the SERS-active sites on Ag electrodes

    NASA Astrophysics Data System (ADS)

    Watanabe, T.; Kawanami, O.; Honda, K.; Pettinger, B.

    1983-12-01

    Evidence is given that SERS-active sites at Ag electrodes are associated with Ag + ions, forming sparingly soluble surface complexes with ligands such as pyridine molecules and halide ions. Such surface Ag + complexes contribute a factor of >800 to the overall (10 7-fold) enhancement, possibly via a resonance Raman effect.

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

    ERIC Educational Resources Information Center

    Roth, Charles E.; Lockwood, Linda G.

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

  17. 78 FR 18576 - Agency Information Collection Activities; Comment Request; Experimental Sites Data Collection...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-27

    ... Agency Information Collection Activities; Comment Request; Experimental Sites Data Collection Instrument... information collection requirements and provide the requested data in the desired format. ED is soliciting... collection instrument will be used to collect specific information/performance data for analysis of...

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

    PubMed Central

    Blain, Amy E.; Mandal, Sema; Wu, Henry; MacNeil, Jessica R.; Harrison, Lee H.; Farley, Monica M.; Lynfield, Ruth; Miller, Lisa; Nichols, Megin; Petit, Sue; Reingold, Arthur; Schaffner, William; Thomas, Ann; Zansky, Shelley M.; Anderson, Raydel; Harcourt, Brian H.; Mayer, Leonard W.; Clark, Thomas A.; Cohn, Amanda C.

    2016-01-01

    In 2009, in the Active Bacterial Core surveillance sites, penicillin was not commonly used to treat meningococcal disease. This is likely because of inconsistent availability of antimicrobial susceptibility testing and ease of use of third-generation cephalosporins. Consideration of current practices may inform future meningococcal disease management guidelines. PMID:27704009

  19. The Thumbs Up Ecology Curriculum: A Fun Group of School Site Activities for Sixth Graders.

    ERIC Educational Resources Information Center

    Smith, John; And Others

    This guide is a collection of "fun" school site activities for sixth graders. Some of the topics covered are: animals, trees, energy and lifestyle, land use and you, energy conservation, and car-pooling. Each section offers both introductory information about the topic as well as questions to ponder such as what, so what, now what, and another way…

  20. 78 FR 33908 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-05

    ... identified Wind Energy Area (WEA) on the OCS offshore Rhode Island (RI) and Massachusetts (MA). The revised... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on the.... BOEM may issue one or more commercial wind energy leases in the WEA. The competitive lease process...

  1. 77 FR 3460 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-24

    ... published a final rule under 10 CFR Part 765 in the Federal Register on May 23, 1994, (59 FR 26714) to carry... for reimbursement. DOE amended the final rule on June 3, 2003, (68 FR 32955) to adopt several... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department...

  2. 75 FR 71677 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-24

    ... published a final rule under 10 CFR Part 765 in the Federal Register on May 23, 1994, (59 FR 26714) to carry... for reimbursement. DOE amended the final rule on June 3, 2003, (68 FR 32955) to adopt several... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department...

  3. Archaeological Activity Report: Post-Review Discoveries Within 45BN431 at Solid Waste Site 128-F-2

    SciTech Connect

    T. E. Marceau; J. J. Sharpe

    2006-12-21

    During monitoring of remedial activities at Solid Waste Site 128-F-2 on August 19, 2005, a concentration of mussel shell was discovered in the west wall of a trench in the northen section of the waste site.

  4. Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

    PubMed

    Taylor, John C; Markham, George D

    2003-07-15

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the

  5. Spectroscopic studies of single and double variants of M ferritin: lack of conversion of a biferrous substrate site into a cofactor site for O2 activation.

    PubMed

    Kwak, Yeonju; Schwartz, Jennifer K; Haldar, Suranjana; Behera, Rabindra K; Tosha, Takehiko; Theil, Elizabeth C; Solomon, Edward I

    2014-01-28

    Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O2 to react with substrate. Ferritin has an active site ligand set with 1-His/4-carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O2 or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O2 activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a μ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O2 reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O2 by the binuclear non-heme iron enzymes.

  6. Atomic structure of anthrax protective antigen pore elucidates toxin translocation.

    PubMed

    Jiang, Jiansen; Pentelute, Bradley L; Collier, R John; Zhou, Z Hong

    2015-05-28

    Anthrax toxin, comprising protective antigen, lethal factor, and oedema factor, is the major virulence factor of Bacillus anthracis, an agent that causes high mortality in humans and animals. Protective antigen forms oligomeric prepores that undergo conversion to membrane-spanning pores by endosomal acidification, and these pores translocate the enzymes lethal factor and oedema factor into the cytosol of target cells. Protective antigen is not only a vaccine component and therapeutic target for anthrax infections but also an excellent model system for understanding the mechanism of protein translocation. On the basis of biochemical and electrophysiological results, researchers have proposed that a phi (Φ)-clamp composed of phenylalanine (Phe)427 residues of protective antigen catalyses protein translocation via a charge-state-dependent Brownian ratchet. Although atomic structures of protective antigen prepores are available, how protective antigen senses low pH, converts to active pore, and translocates lethal factor and oedema factor are not well defined without an atomic model of its pore. Here, by cryo-electron microscopy with direct electron counting, we determine the protective antigen pore structure at 2.9-Å resolution. The structure reveals the long-sought-after catalytic Φ-clamp and the membrane-spanning translocation channel, and supports the Brownian ratchet model for protein translocation. Comparisons of four structures reveal conformational changes in prepore to pore conversion that support a multi-step mechanism by which low pH is sensed and the membrane-spanning channel is formed.

  7. Atomic structure of anthrax PA pore elucidates toxin translocation

    PubMed Central

    Jiang, Jiansen; Pentelute, Bradley L.; Collier, R. John; Zhou, Z. Hong

    2015-01-01

    Summary Anthrax toxin, comprising protective antigen (PA), lethal factor (LF) and edema factor (EF), is the major virulence factor of Bacillus anthracis, an agent that causes high mortality in human and animals. PA forms oligomeric prepores that undergo conversion to membrane-spanning pores by endosomal acidification, and these pores translocate the enzymes LF and EF into the cytosol of target cells1. PA is not only a vaccine component and therapeutic target for anthrax infections but also an excellent model system for understanding the mechanism of protein translocation. Based on biochemical and electrophysiological results, researchers have proposed that a Φ-clamp composed of Phe427 residues of PA catalyzes protein translocation via a charge-state dependent Brownian ratchet2–9. Although atomic structures of PA prepores are available10–14, how PA senses low pH, converts to active pore and translocates LF and EF are not well defined without an atomic model of the PA pore. Here, by cryo electron microscopy (cryoEM) with direct electron counting, we have determined the PA pore structure at 2.9-Å resolution. The structure reveals the long-sought-after catalytic Φ-clamp and the membrane-spanning translocation channel, and supports the Brownian ratchet model for protein translocation. Comparisons of four structures reveal conformational changes in prepore to pore conversion that support a multi-step mechanism by which low-pH is sensed and the membrane-spanning channel is formed. PMID:25778700

  8. DESIGN INFORMATION ON FINE PORE AERATION SYSTEMS

    EPA Science Inventory

    Field studies were conducted over several years at municipal wastewater treatment plants employing line pore diffused aeration systems. These studies were designed to produce reliable information on the performance and operational requirements of fine pore devices under process ...

  9. Characterization of the active site properties of CYP4F12.

    PubMed

    Eksterowicz, John; Rock, Dan A; Rock, Brooke M; Wienkers, Larry C; Foti, Robert S

    2014-10-01

    Cytochrome P450 4F12 is a drug-metabolizing enzyme that is primarily expressed in the liver, kidney, colon, small intestine, and heart. The properties of CYP4F12 that may impart an increased catalytic selectivity (decreased promiscuity) were explored through in vitro metabolite elucidation, kinetic isotope effect experiments, and computational modeling of the CYP4F12 active site. By using astemizole as a probe substrate for CYP4F12 and CYP3A4, it was observed that although CYP4F12 favored astemizole O-demethylation as the primary route of metabolism, CYP3A4 was capable of metabolizing astemizole at multiple sites on the molecule. Deuteration of astemizole at the site of O-demethylation resulted in an isotope effect of 7.1 as well as an 8.3-fold decrease in the rate of clearance for astemizole by CYP4F12. Conversely, although an isotope effect of 3.8 was observed for the formation of the O-desmethyl metabolite when deuterated astemizole was metabolized by CYP3A4, there was no decrease in the clearance of astemizole. Development of a homology model of CYP4F12 based on the crystal structure of cytochrome P450 BM3 predicted an active site volume for CYP4F12 that was approximately 76% of the active site volume of CYP3A4. As predicted, multiple favorable binding orientations were available for astemizole docked into the active site of CYP3A4, but only a single binding orientation with the site of O-demethylation oriented toward the heme was identified for CYP4F12. Overall, it appears that although CYP4F12 may be capable of binding similar ligands to other cytochrome P450 enzymes such as CYP3A4, the ability to achieve catalytically favorable orientations may be inherently more difficult because of the increased steric constraints of the CYP4F12 active site. PMID:25074871

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

    PubMed Central

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

    1999-01-01

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

  11. Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase

    SciTech Connect

    Yip, Wing-Kin; Dong, Jian-Guo; Yang, S.F. ); Kenny, J.W.; Thompson, G.A. )

    1990-10-01

    The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB{sup 3}H{sub 4} or Ado({sup 14}C)Met. Peptide sequencing of both {sup 3}H- and {sup 14}C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the {sup 3}H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the {sup 14}C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado ({sup 14}C)Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6.

  12. Extending electromagnetic methods to map coastal pore water salinities

    USGS Publications Warehouse

    Greenwood, Wm. J.; Kruse, S.; Swarzenski, P.

    2006-01-01

    The feasibility of mapping pore water salinity based on surface electromagnetic (EM) methods over land and shallow marine water is examined in a coastal wetland on Tampa Bay, Florida. Forward models predict that useful information on seabed conductivity can be obtained through <1.5 m of saline water, using floating EM-31 and EM-34 instruments from Geonics Ltd. The EM-31 functioned as predicted when compared against resistivity soundings and pore water samples and proved valuable for profiling in otherwise inaccessible terrain due to its relatively small size. Experiments with the EM-34 in marine water, however, did not reproduce the theoretical instrument response. The most effective technique for predicting pore water conductivities based on EM data entailed (1) computing formation factors from resistivity surveys and pore water samples at representative sites and (2) combining these formation factors with onshore and offshore EM-31 readings for broader spatial coverage. This method proved successful for imaging zones of elevated pore water conductivities/ salinities associated with mangrove forests, presumably caused by salt water exclusion by mangrove roots. These zones extend 5 to 10 m seaward from mangrove trunks fringing Tampa Bay. Modeling indicates that EM-31 measurements lack the resolution necessary to image the subtle pore water conductivity variations expected in association with diffuse submarine ground water discharge of fresher water in the marine water of Tampa Bay. The technique has potential for locating high-contrast zones and other pore water salinity anomalies in areas not accessible to conventional marine- or land-based resistivity arrays and hence may be useful for studies of coastal-wetland ecosystems. Copyright ?? 2005 National Ground Water Association.

  13. Lymphokine-activated killer (LAK) cells can be focused at sites of tumor growth by products of macrophage activation

    SciTech Connect

    Migliori, R.J.; Gruber, S.A.; Sawyer, M.D.; Hoffman, R.; Ochoa, A.; Bach, F.H.; Simmons, R.L.

    1987-08-01

    Successful adoptive cancer immunotherapy presumably depends on the accumulation of tumoricidal leukocytes at the sites of tumor growth. Large numbers of lymphokine-activated killer (LAK) cells can be generated in vitro by growth in high concentrations of interleukin-2 (IL-2), but relatively few arrive at the tumor site after intravenous injection. We hypothesize that the delivery of LAK cells to tumor sites may be augmented by previously demonstrated lymphocyte-recruiting factors, including activated macrophage products such as interleukin-1 (IL-1) and tumor necrosis factor. /sup 111/Indium-labeled LAK cells were injected intravenously into syngeneic mice bearing the macrophage activator endotoxin (LPS) in one hind footpad, and saline solution was injected into the contralateral footpad. Significantly more activity was recovered from the LPS-bearing footpad at all times during a 96-hour period. Recombinant IL-1 also attracted more LAK cells after injection into tumor-free hind footpads. Furthermore, LAK cells preferentially homed to hind footpads that were bearing 3-day established sarcomas after intralesional injections of LPS, IL-1, or tumor necrosis factor when compared with contralateral tumor-bearing footpads injected with saline solution alone. In preliminary experiments, mice with hind-footpad tumors appeared to survive longer after combined systemic IL-2 and LAK therapy if intralesional LPS was administered. These studies demonstrate that macrophage activation factors that have been shown capable of attracting circulating normal lymphocytes can also effectively attract LAK cells from the circulation. By the stimulation of macrophages at the sites of tumor growth, more LAK cells can be attracted. It is hoped that by focusing the migration of LAK cells to tumors, LAK cells and IL-2 would effect tumor regression more efficiently and with less toxicity.

  14. 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

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

    PubMed Central

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

    2014-01-01

    The ability to make behavioural inferences from skeletal remains is critical to understanding the lifestyles and activities of past human populations and extinct animals. Muscle attachment site (enthesis) morphology has long been assumed to reflect muscle strength and activity during life, but little experimental evidence exists to directly link activity patterns with muscle development and the morphology of their attachments to the skeleton. We used a mouse model to experimentally test how the level and type of activity influences forelimb muscle architecture of spinodeltoideus, acromiodeltoideus, and superficial pectoralis, bone growth rate and gross morphology of their insertion sites. Over an 11-week period, we collected data on activity levels in one control group and two experimental activity groups (running, climbing) of female wild-type mice. Our results show that both activity type and level increased bone growth rates influenced muscle architecture, including differences in potential muscular excursion (fibre length) and potential force production (physiological cross-sectional area). However, despite significant influences on muscle architecture and bone development, activity had no observable effect on enthesis morphology. These results suggest that the gross morphology of entheses is less reliable than internal bone structure for making inferences about an individual’s past behaviour. PMID:25467113

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

    PubMed

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

    2007-09-01

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

  17. Asymmetry of the active site loop conformation between subunits of glutamate-1-semialdehyde aminomutase in solution.

    PubMed

    Campanini, Barbara; Bettati, Stefano; di Salvo, Martino Luigi; Mozzarelli, Andrea; Contestabile, Roberto

    2013-01-01

    Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5'-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5'-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.

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

    PubMed Central

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

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

  19. Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

    PubMed

    Gross, M; Whetzel, N K; Folk, J E

    1975-10-10

    Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate. PMID:240837

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

    PubMed

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

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

  1. Alternative poly(A) site utilization during adenovirus infection coincides with a decrease in the activity of a poly(A) site processing factor.

    PubMed Central

    Mann, K P; Weiss, E A; Nevins, J R

    1993-01-01

    The recognition and processing of a pre-mRNA to create a poly(A) addition site, a necessary step in mRNA biogenesis, can also be a regulatory event in instances in which the frequency of use of a poly(A) site varies. One such case is found during the course of an adenovirus infection. Five poly(A) sites are utilized within the major late transcription unit to produce more than 20 distinct mRNAs during the late phase of infection. The proximal half of the major late transcription unit is also expressed during the early phase of a viral infection. During this early phase of expression, the L1 poly(A) site is used three times more frequently than the L3 poly(A) site. In contrast, the L3 site is used three times more frequently than the L1 site during the late phase of infection. Recent experiments have suggested that the recognition of the poly(A) site GU-rich downstream element by the CF1 processing factor may be a rate-determining step in poly(A) site selection. We demonstrate that the interaction of CF1 with the L1 poly(A) site is less stable than the interaction of CF1 with the L3 poly(A) site. We also find that there is a substantial decrease in the level of CF1 activity when an adenovirus infection proceeds to the late phase. We suggest that this reduction in CF1 activity, coupled with the relative instability of the interaction with the L1 poly(A) site, contributes to the reduced use of the L1 poly(A) site during the late stage of an adenovirus infection. Images PMID:8384308

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

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

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

    PubMed

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

    2016-04-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

  4. Estimating methane production rates in bogs and landfills by deuterium enrichment of pore water

    USGS Publications Warehouse

    Siegel, D.I.; Chanton, J.P.; Glaser, P.H.; Chasar, L.S.; Rosenberry, D.O.

    2001-01-01

    Raised bogs and municipal waste landfills harbor large populations of methanogens within their domed deposits of anoxic organic matter. Although the methane emissions from these sites have been estimated by various methods, limited data exist on the activity of the methanogens at depth. We therefore analyzed the stable isotopic signature of the pore waters in two raised bogs from northern Minnesota to identify depth intervals in the peat profile where methanogenic metabolism occurs. Methanogenesis enriched the deuterium (2H) content of the deep peat pore waters by as much as +11% (Vienna Standard Mean Sea Water), which compares to a much greater enrichment factor of +70% in leachate from New York City's Fresh Kills landfill. The bog pore waters were isotopically dated by tritium (3H) to be about 35 years old at 1.5 m depth, whereas the landfill leachate was estimated as ~ 17 years old from Darcy flow calculations. According to an isotopic mass balance the observed deuterium enrichment indicates that about 1.2 g of CH4m-3 d-1 were produced within the deeper peat, compared to about 2.8 g CH4 m-3 d-1 in the landfill. The values for methane production in the bog peat are substantially higher than the flux rates measured at the surface of the bogs or at the landfill, indicating that deeper methane production may be much higher than was previously assumed.

  5. Estimating methane production rates in bogs and landfills by deuterium enrichment of pore water

    NASA Astrophysics Data System (ADS)

    Siegel, D. I.; Chanton, J. R.; Glaser, P. H.; Chasar, L. S.; Rosenberry, D. O.

    2001-12-01

    Raised bogs and municipal waste landfills harbor large populations of methanogens within their domed deposits of anoxic organic matter. Although the methane emissions from these sites have been estimated by various methods, limited data exist on the activity of the methanogens at depth. We therefore analyzed the stable isotopic signature of the pore waters in two raised bogs from northern Minnesota to identify depth intervals in the peat profile where methanogenic metabolism occurs. Methanogenesis enriched the deuterium (2H) content of the deep peat pore waters by as much as +11‰ (Vienna Standard Mean Sea Water), which compares to a much greater enrichment factor of +70‰ in leachate from New York City's Fresh Kills landfill. The bog pore waters were isotopically dated by tritium (3H) to be about 35 years old at 1.5 m depth, whereas the landfill leachate was estimated as ˜17 years old from Darcy flow calculations. According to an isotopic mass balance the observed deuterium enrichment indicates that about 1.2 g of CH4 m-3 d-1 were produced within the deeper peat, compared to about 2.8 g CH4 m-3 d-1 in the landfill. The values for methane production in the bog peat are substantially higher than the flux rates measured at the surface of the bogs or at the landfill, indicating that deeper methane production may be much higher than was previously assumed.

  6. Antimicrobial Peptides in Toroidal and Cylindrical Pores

    PubMed Central

    Mihajlovic, Maja

    2010-01-01

    Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize biological membranes. Their mechanism of action is still not well understood. Here we investigate the preference of alamethicin and melittin for pores of different shapes, using molecular dynamics (MD) simulations of the peptides in pre-formed toroidal and cylindrical pores. When an alamethicin hexamer is initially embedded in a cylindrical pore, at the end of the simulation the pore remains cylindrical or closes if glutamines in the N-termini are not located within the pore. On the other hand, when a melittin tetramer is embedded in toroidal pore or in a cylindrical pore, at the end of the simulation the pore is lined both with peptides and lipid headgroups, and, thus, can be classified as a toroidal pore. These observations agree with the prevailing views that alamethicin forms barrel-stave pores whereas melittin forms toroidal pores. Both alamethicin and melittin form amphiphilic helices in the presence of membranes, but their net charge differs; at pH ~7, the net charge of alamethicin is −1 whereas that of melittin is +5. This gives rise to stronger electrostatic interactions of melittin with membranes than those of alamethicin. The melittin tetramer interacts more strongly with lipids in the toroidal pore than in the cylindrical one, due to more favorable electrostatic interactions. PMID:20403332

  7. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    SciTech Connect

    Parker, Shane M.; Shiozaki, Toru