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Sample records for nickel-loaded protein-affinity selection

  1. Nickel nanoparticle decorated graphene for highly selective isolation of polyhistidine-tagged proteins

    Science.gov (United States)

    Liu, Jia-Wei; Yang, Ting; Ma, Lin-Yu; Chen, Xu-Wei; Wang, Jian-Hua

    2013-12-01

    Nickel nanoparticle decorated graphene (GP-Ni) is prepared by one-pot hydrothermal reduction of graphene oxide and nickel cations by hydrazine hydrate in the presence of poly(sodium-p-styrenesulfonate) (PSS). The GP-Ni hybrid is characterized by XRD, TEM, SEM, XPS, Raman and FT-IR spectra, demonstrating the formation of poly-dispersed nickel nanoparticles with an average size of 83 nm attached on the surface of graphene sheets. The GP-Ni hybrid exhibits ferromagnetic behavior with a magnetization saturation of 31.1 emu g-1 at 10 000 Oersted (Oe). The GP-Ni also possesses favorable stability in aqueous medium and rapid magnetic response to an external magnetic field. These make it a novel magnetic adsorbent for the separation/isolation of His6-tagged recombinant proteins from a complex sample matrix (cell lysate). The targeted protein species is captured onto the surface of the GP-Ni hybrid via specific metal affinity force between polyhistidine groups and nickel nanoparticles. The SDS-PAGE assay indicates highly selective separation of His6-tagged Smt A from cell lysate. The GP-Ni hybrid displays favorable performance on the separation/isolation of His6-tagged recombinant proteins with respect to the commercial NTA-Ni2+ column.

  2. Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

    Science.gov (United States)

    Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

    2015-09-01

    Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Structural Basis of Low-Affinity Nickel Binding to the Nickel-Responsive Transcription Factor NikR from Escherichia coli

    International Nuclear Information System (INIS)

    Phillips, C.; Schreiter, E.; Stultz, C.; Drennan, C.

    2010-01-01

    Escherichia coli NikR regulates cellular nickel uptake by binding to the nik operon in the presence of nickel and blocking transcription of genes encoding the nickel uptake transporter. NikR has two binding affinities for the nik operon: a nanomolar dissociation constant with stoichiometric nickel and a picomolar dissociation constant with excess nickel (Bloom, S. L., and Zamble, D. B. (2004) Biochemistry 43, 10029-10038; Chivers, P. T., and Sauer, R. T. (2002) Chem. Biol. 9, 1141-1148). While it is known that the stoichiometric nickel ions bind at the NikR tetrameric interface (Schreiter, E. R., et al. (2003) Nat. Struct. Biol. 10, 794-799; Schreiter, E. R., et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 13676-13681), the binding sites for excess nickel ions have not been fully described. Here we have determined the crystal structure of NikR in the presence of excess nickel to 2.6 (angstrom) resolution and have obtained nickel anomalous data (1.4845 (angstrom)) in the presence of excess nickel for both NikR alone and NikR cocrystallized with a 30-nucleotide piece of double-stranded DNA containing the nik operon. These anomalous data show that excess nickel ions do not bind to a single location on NikR but instead reveal a total of 22 possible low-affinity nickel sites on the NikR tetramer. These sites, for which there are six different types, are all on the surface of NikR, and most are found in both the NikR alone and NikR-DNA structures. Using a combination of crystallographic data and molecular dynamics simulations, the nickel sites can be described as preferring octahedral geometry, utilizing one to three protein ligands (typically histidine) and at least two water molecules.

  4. A protein engineered to bind uranyl selectively and with femtomolar affinity

    Science.gov (United States)

    Zhou, Lu; Bosscher, Mike; Zhang, Changsheng; Özçubukçu, Salih; Zhang, Liang; Zhang, Wen; Li, Charles J.; Liu, Jianzhao; Jensen, Mark P.; Lai, Luhua; He, Chuan

    2014-03-01

    Uranyl (UO22+), the predominant aerobic form of uranium, is present in the ocean at a concentration of ~3.2 parts per 109 (13.7 nM) however, the successful enrichment of uranyl from this vast resource has been limited by the high concentrations of metal ions of similar size and charge, which makes it difficult to design a binding motif that is selective for uranyl. Here we report the design and rational development of a uranyl-binding protein using a computational screening process in the initial search for potential uranyl-binding sites. The engineered protein is thermally stable and offers very high affinity and selectivity for uranyl with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions. We also demonstrated that the uranyl-binding protein can repeatedly sequester 30-60% of the uranyl in synthetic sea water. The chemical strategy employed here may be applied to engineer other selective metal-binding proteins for biotechnology and remediation applications.

  5. Affinity selection-mass spectrometry and its emerging application to the high throughput screening of G protein-coupled receptors.

    Science.gov (United States)

    Whitehurst, Charles E; Annis, D Allen

    2008-07-01

    Advances in combinatorial chemistry and genomics have inspired the development of novel affinity selection-based screening techniques that rely on mass spectrometry to identify compounds that preferentially bind to a protein target. Of the many affinity selection-mass spectrometry techniques so far documented, only a few solution-based implementations that separate target-ligand complexes away from unbound ligands persist today as routine high throughput screening platforms. Because affinity selection-mass spectrometry techniques do not rely on radioactive or fluorescent reporters or enzyme activities, they can complement traditional biochemical and cell-based screening assays and enable scientists to screen targets that may not be easily amenable to other methods. In addition, by employing mass spectrometry for ligand detection, these techniques enable high throughput screening of massive library collections of pooled compound mixtures, vastly increasing the chemical space that a target can encounter during screening. Of all drug targets, G protein coupled receptors yield the highest percentage of therapeutically effective drugs. In this manuscript, we present the emerging application of affinity selection-mass spectrometry to the high throughput screening of G protein coupled receptors. We also review how affinity selection-mass spectrometry can be used as an analytical tool to guide receptor purification, and further used after screening to characterize target-ligand binding interactions, enabling the classification of orthosteric and allosteric binders.

  6. Engineering of bispecific affinity proteins with high affinity for ERBB2 and adaptable binding to albumin.

    Directory of Open Access Journals (Sweden)

    Johan Nilvebrant

    Full Text Available The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.

  7. Specific capture of uranyl protein targets by metal affinity chromatography

    International Nuclear Information System (INIS)

    Basset, C.; Dedieu, A.; Guerin, P.; Quemeneur, E.; Meyer, D.; Vidaud, C.

    2008-01-01

    To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography (IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO 2 2+ ) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of amino-phosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis. (authors)

  8. High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

    International Nuclear Information System (INIS)

    Orito, N; Umekage, S; Sakai, E; Tanaka, T; Kikuchi, Y; Sato, K; Kawauchi, S; Tanaka, H

    2012-01-01

    We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be K D = 2.25x10 -9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

  9. Specificity and affinity quantification of protein-protein interactions.

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    Yan, Zhiqiang; Guo, Liyong; Hu, Liang; Wang, Jin

    2013-05-01

    Most biological processes are mediated by the protein-protein interactions. Determination of the protein-protein structures and insight into their interactions are vital to understand the mechanisms of protein functions. Currently, compared with the isolated protein structures, only a small fraction of protein-protein structures are experimentally solved. Therefore, the computational docking methods play an increasing role in predicting the structures and interactions of protein-protein complexes. The scoring function of protein-protein interactions is the key responsible for the accuracy of the computational docking. Previous scoring functions were mostly developed by optimizing the binding affinity which determines the stability of the protein-protein complex, but they are often lack of the consideration of specificity which determines the discrimination of native protein-protein complex against competitive ones. We developed a scoring function (named as SPA-PP, specificity and affinity of the protein-protein interactions) by incorporating both the specificity and affinity into the optimization strategy. The testing results and comparisons with other scoring functions show that SPA-PP performs remarkably on both predictions of binding pose and binding affinity. Thus, SPA-PP is a promising quantification of protein-protein interactions, which can be implemented into the protein docking tools and applied for the predictions of protein-protein structure and affinity. The algorithm is implemented in C language, and the code can be downloaded from http://dl.dropbox.com/u/1865642/Optimization.cpp.

  10. Spectral affinity in protein networks.

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    Voevodski, Konstantin; Teng, Shang-Hua; Xia, Yu

    2009-11-29

    Protein-protein interaction (PPI) networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise than simpler methods based on edge density and shortest path length. We develop a novel affinity measure for pairs of proteins in PPI networks, which uses personalized PageRank, a random walk based method used in context-sensitive search on the Web. Our measure of closeness, which we call PageRank Affinity, is proportional to the number of times the smaller-degree protein is visited in a random walk that restarts at the larger-degree protein. PageRank considers paths of all lengths in a network, therefore PageRank Affinity is a precise measure that is robust to noise in the data. PageRank Affinity is also provably related to cluster co-membership, making it a meaningful measure. In our experiments on protein networks we find that our measure is better at predicting co-complex membership and finding functionally related proteins than other commonly used measures of closeness. Moreover, our experiments indicate that PageRank Affinity is very resilient to noise in the network. In addition, based on our method we build a tool that quickly finds nodes closest to a queried protein in any protein network, and easily scales to much larger biological networks. We define a meaningful way to assess the closeness of two proteins in a PPI network, and show that our closeness measure is more biologically significant than other commonly used methods. We also develop a tool, accessible at http://xialab.bu.edu/resources/pnns, that allows the user to quickly find nodes closest to a queried vertex in any protein

  11. Super magnetic nanoparticles NiFe2O4, coated with aluminum-nickel oxide sol-gel lattices to safe, sensitive and selective purification of his-tagged proteins.

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    Mirahmadi-Zare, Seyede Zohreh; Allafchian, Alireza; Aboutalebi, Fatemeh; Shojaei, Pendar; Khazaie, Yahya; Dormiani, Kianoush; Lachinani, Liana; Nasr-Esfahani, Mohammad-Hossein

    2016-05-01

    Super magnetic nanoparticle NiFe2O4 with high magnetization, physical and chemical stability was introduced as a core particle which exhibits high thermal stability (>97%) during the harsh coating process. Instead of multi-stage process for coating, the magnetic nanoparticles was mineralized via one step coating by a cheap, safe, stable and recyclable alumina sol-gel lattice (from bohemite source) saturated by nickel ions. The TEM, SEM, VSM and XRD imaging and BET analysis confirmed the structural potential of NiFe2O4@NiAl2O4 core-shell magnetic nanoparticles for selective and sensitive purification of His-tagged protein, in one step. The functionality and validity of the nickel magnetic nanoparticles were attested by purification of three different bioactive His-tagged recombinant fusion proteins including hIGF-1, GM-CSF and bFGF. The bonding capacity of the nickel magnetics nanoparticles was studied by Bradford assay and was equal to 250 ± 84 μg Protein/mg MNP base on protein size. Since the metal ion leakage is the most toxicity source for purification by nickel magnetic nanoparticles, therefor the nickel leakage in purified final protein was determined by atomic absorption spectroscopy and biological activity of final purified protein was confirmed in comparison with reference. Also, in vitro cytotoxicity of nickel magnetic nanoparticles and trace metal ions were investigated by MTS assay analysis. The results confirmed that the synthesized nickel magnetic nanoparticles did not show metal ion toxicity and not affected on protein folding. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

    Science.gov (United States)

    SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

    2016-01-01

    SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

  13. Spectral affinity in protein networks

    Directory of Open Access Journals (Sweden)

    Teng Shang-Hua

    2009-11-01

    Full Text Available Abstract Background Protein-protein interaction (PPI networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise than simpler methods based on edge density and shortest path length. Results We develop a novel affinity measure for pairs of proteins in PPI networks, which uses personalized PageRank, a random walk based method used in context-sensitive search on the Web. Our measure of closeness, which we call PageRank Affinity, is proportional to the number of times the smaller-degree protein is visited in a random walk that restarts at the larger-degree protein. PageRank considers paths of all lengths in a network, therefore PageRank Affinity is a precise measure that is robust to noise in the data. PageRank Affinity is also provably related to cluster co-membership, making it a meaningful measure. In our experiments on protein networks we find that our measure is better at predicting co-complex membership and finding functionally related proteins than other commonly used measures of closeness. Moreover, our experiments indicate that PageRank Affinity is very resilient to noise in the network. In addition, based on our method we build a tool that quickly finds nodes closest to a queried protein in any protein network, and easily scales to much larger biological networks. Conclusion We define a meaningful way to assess the closeness of two proteins in a PPI network, and show that our closeness measure is more biologically significant than other commonly used methods. We also develop a tool, accessible at http://xialab.bu.edu/resources/pnns, that allows the user to

  14. A new affinity-HPLC packing for protein separation: Cibacron blue attached uniform porous poly(HEMA-co-EDM) beads.

    Science.gov (United States)

    Unsal, Ender; Durdu, Aysun; Elmas, Begum; Tuncel, Murvet; Tuncel, Ali

    2005-11-01

    In this study, a new affinity high-performance liquid chromatography (HPLC) stationary phase suitable for protein separation was synthesized. In the first stage of the synthesis, uniform porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(HEMA-co-EDM), beads 6.2 mum in size were obtained. Homogeneous distribution of hydroxyl groups in the bead interior was confirmed by confocal laser scanning microscopy. The plain poly(HEMA-co-EDM) particles gave very low non-specific protein adsorption with albumin. The selected dye ligand Cibacron blue F3G-A (CB F3G-A) was covalently linked onto the beads via hydroxyl groups. In the batch experiments, albumin adsorption up to 60 mg BSA/g particles was obtained with the CB F3G-A carrying poly(HEMA-co-EDM) beads. The affinity-HPLC of selected proteins (albumin and lysozyme) was investigated in a 25 mm x 4.0-mm inner diameter column packed with CB F3G-A carrying beads and both proteins were successfully resolved. By a single injection, 200 mug of protein was loaded and quantitatively eluted from the column. The protein recovery increased with increasing flow rate and salt concentration of the elution buffer and decreased with the increasing protein feed concentration. During the albumin elution, theoretical plate numbers up to 30,000 plates/m were achieved by increasing the salt concentration.

  15. Influence of ni addition to a low-loaded palladium catalyst on the selective hydrogenation of 1-heptyne

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    Cecilia R. Lederhos

    2010-01-01

    Full Text Available Semi-hydrogenation of alkynes has industrial and academic relevance on a large scale. To increase the activity, selectivity and lifetime of monometallic catalysts, the development of bimetallic catalysts has been investigated. 1-Heptyne hydrogenation over low-loaded Pd and Ni monometallic and PdNi bimetallic catalysts was studied in liquid phase at mild conditions. XPS results suggest that nickel addition to Pd modifies the electronic state of palladium as nickel loading is increased. Low-loaded Pd catalysts showed the highest selectivities (> 95%. The most active prepared catalyst, PdNi(1%, was more selective than the Lindlar catalyst.

  16. Fragment-based quantum mechanical calculation of protein-protein binding affinities.

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    Wang, Yaqian; Liu, Jinfeng; Li, Jinjin; He, Xiao

    2018-04-29

    The electrostatically embedded generalized molecular fractionation with conjugate caps (EE-GMFCC) method has been successfully utilized for efficient linear-scaling quantum mechanical (QM) calculation of protein energies. In this work, we applied the EE-GMFCC method for calculation of binding affinity of Endonuclease colicin-immunity protein complex. The binding free energy changes between the wild-type and mutants of the complex calculated by EE-GMFCC are in good agreement with experimental results. The correlation coefficient (R) between the predicted binding energy changes and experimental values is 0.906 at the B3LYP/6-31G*-D level, based on the snapshot whose binding affinity is closest to the average result from the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculation. The inclusion of the QM effects is important for accurate prediction of protein-protein binding affinities. Moreover, the self-consistent calculation of PB solvation energy is required for accurate calculations of protein-protein binding free energies. This study demonstrates that the EE-GMFCC method is capable of providing reliable prediction of relative binding affinities for protein-protein complexes. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

  17. Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

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    Hayashi, Takahiro; Hamachi, Itaru

    2012-09-18

    Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

  18. Accurate and sensitive quantification of protein-DNA binding affinity.

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    Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

    2018-04-17

    Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

  19. Development of novel nanocomposite adsorbent based on potassium nickel hexacyanoferrate-loaded polypropylene fabric.

    Science.gov (United States)

    Bondar, Yuliia; Kuzenko, Svetlana; Han, Do-Hung; Cho, Hyun-Kug

    2014-01-01

    A nanocomposite adsorbent based on potassium nickel hexacyanoferrate-loaded polypropylene fabric was synthesized for selective removal of Cs ions from contaminated waters by a two-stage synthesis: radiation-induced graft polymerization of acrylic acid monomer onto the nonwoven polypropylene fabric surface with subsequent in situ formation of potassium nickel hexacyanoferrate (KNiHCF) nanoparticles within the grafted chains. Data of scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy confirmed the formation of KNiHCF homogeneous phase on the fabric surface, which consisted of crystalline cubic-shaped nanoparticles (70 to 100 nm). The efficiency of the synthesized adsorbent for removal of cesium ions was evaluated under various experimental conditions. It has demonstrated a rapid adsorption process, high adsorption capacity over a wide pH range, and selectivity in Cs ion removal from model solutions with high concentration of sodium ions.

  20. Extraordinary Diversity of Immune Response Proteins among Sea Urchins: Nickel-Isolated Sp185/333 Proteins Show Broad Variations in Size and Charge

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    Sherman, Lauren S.; Schrankel, Catherine S.; Brown, Kristy J.; Smith, L. Courtney

    2015-01-01

    Effective protection against pathogens requires the host to produce a wide range of immune effector proteins. The Sp185/333 gene family, which is expressed by the California purple sea urchin Strongylocentrotus purpuratus in response to bacterial infection, encodes a highly diverse repertoire of anti-pathogen proteins. A subset of these proteins can be isolated by affinity to metal ions based on multiple histidines, resulting in one to four bands of unique molecular weight on standard Western blots, which vary depending on the individual sea urchin. Two dimensional gel electrophoresis (2DE) of nickel-isolated protein samples followed by Western blot was employed to detect nickel-isolated Sp185/333 (Ni-Sp185/333) proteins and to evaluate protein diversity in animals before and after immune challenge with marine bacteria. Ni-Sp185/333 proteins of the same molecular weight on standard Western blots appear as a broad complex of variants that differ in pI on 2DE Western blots. The Ni-Sp185/333 protein repertoire is variable among animals, and shows a variety of changes among individual sea urchins in response to immune challenges with both the same and different species of bacteria. The extraordinary diversity of the Ni-Sp185/333 proteins may provide significant anti-pathogen capabilities for sea urchins that survive solely on innate immunity. PMID:26406912

  1. Learning a peptide-protein binding affinity predictor with kernel ridge regression

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    2013-01-01

    Background The cellular function of a vast majority of proteins is performed through physical interactions with other biomolecules, which, most of the time, are other proteins. Peptides represent templates of choice for mimicking a secondary structure in order to modulate protein-protein interaction. They are thus an interesting class of therapeutics since they also display strong activity, high selectivity, low toxicity and few drug-drug interactions. Furthermore, predicting peptides that would bind to a specific MHC alleles would be of tremendous benefit to improve vaccine based therapy and possibly generate antibodies with greater affinity. Modern computational methods have the potential to accelerate and lower the cost of drug and vaccine discovery by selecting potential compounds for testing in silico prior to biological validation. Results We propose a specialized string kernel for small bio-molecules, peptides and pseudo-sequences of binding interfaces. The kernel incorporates physico-chemical properties of amino acids and elegantly generalizes eight kernels, comprised of the Oligo, the Weighted Degree, the Blended Spectrum, and the Radial Basis Function. We provide a low complexity dynamic programming algorithm for the exact computation of the kernel and a linear time algorithm for it’s approximation. Combined with kernel ridge regression and SupCK, a novel binding pocket kernel, the proposed kernel yields biologically relevant and good prediction accuracy on the PepX database. For the first time, a machine learning predictor is capable of predicting the binding affinity of any peptide to any protein with reasonable accuracy. The method was also applied to both single-target and pan-specific Major Histocompatibility Complex class II benchmark datasets and three Quantitative Structure Affinity Model benchmark datasets. Conclusion On all benchmarks, our method significantly (p-value ≤ 0.057) outperforms the current state-of-the-art methods at predicting

  2. Selectable high-yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support.

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    Schellenberg, Matthew J; Petrovich, Robert M; Malone, Christine C; Williams, R Scott

    2018-03-25

    Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion-tag to generate recombinant proteins using suspension-cultured HEK293F cells. YFP is a dual-function tag that enables direct visualization and fluorescence-based selection of high expressing clones for and rapid purification using a high-stringency, high-affinity anti-GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression. Published 2018. This article is a US Government work and is in the public domain in the USA.

  3. Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

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    M Lisa Phipps

    Full Text Available Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1 ensure efficient display; 2 maximize the ability to select high affinity ligands; and 3 minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.

  4. Calculation of protein-ligand binding affinities.

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    Gilson, Michael K; Zhou, Huan-Xiang

    2007-01-01

    Accurate methods of computing the affinity of a small molecule with a protein are needed to speed the discovery of new medications and biological probes. This paper reviews physics-based models of binding, beginning with a summary of the changes in potential energy, solvation energy, and configurational entropy that influence affinity, and a theoretical overview to frame the discussion of specific computational approaches. Important advances are reported in modeling protein-ligand energetics, such as the incorporation of electronic polarization and the use of quantum mechanical methods. Recent calculations suggest that changes in configurational entropy strongly oppose binding and must be included if accurate affinities are to be obtained. The linear interaction energy (LIE) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods are analyzed, as are free energy pathway methods, which show promise and may be ready for more extensive testing. Ultimately, major improvements in modeling accuracy will likely require advances on multiple fronts, as well as continued validation against experiment.

  5. Nickel decreases cellular iron level and converts cytosolic aconitase to iron-regulatory protein 1 in A549 cells

    International Nuclear Information System (INIS)

    Chen Haobin; Davidson, Todd; Singleton, Steven; Garrick, Michael D.; Costa, Max

    2005-01-01

    Nickel (Ni) compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia-inducible factor-1 alpha (HIF-1α). This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase (c-aconitase) activity in A549 cells. Cytosolic aconitase was converted to iron-regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. The increased activity of iron-regulatory protein 1 after nickel exposure stabilized and increased transferrin receptor (Tfr) mRNA and antagonized the iron-induced ferritin light chain protein synthesis. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe-4S] cluster reconstitution by nickel. Exposure of A549 cells to soluble nickel decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron-regulatory protein 1 activity. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron-regulatory protein 1. To assess the mechanism for the observed effects, human embryonic kidney (HEK) cells over expressing divalent metal transporter-1 (DMT1) were compared to A549 cells expressing only endogenous transporters for inhibition of iron uptake by nickel. The inhibition data suggest that nickel can enter via DMT1 and compete with iron for entry into the cell. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions, as well as create a state of hypoxia in cells under normal oxygen tension. These effects may be very important in how nickel exerts phenotypic

  6. Selection of imprinted nanoparticles by affinity chromatography.

    Science.gov (United States)

    Guerreiro, António R; Chianella, Iva; Piletska, Elena; Whitcombe, Michael J; Piletsky, Sergey A

    2009-04-15

    Soluble molecularly imprinted nanoparticles were synthesised via iniferter initiated polymerisation and separated by size via gel permeation chromatography. Subsequent fractionation of these particles by affinity chromatography allowed the separation of high affinity fractions from the mixture of nanoparticles. Fractions selected this way possess affinity similar to that of natural antibodies (K(d) 6.6x10(-8)) M and were also able to discriminate between related functional analogues of the template.

  7. Selective leaching studies of deep-sea sediments loaded with americium, neptunium and plutonium

    International Nuclear Information System (INIS)

    Cole, T.G.; Higgo, J.J.W.; Cronan, D.S.; Rees, L.V.C.

    1984-07-01

    A series of selective leaching experiments were undertaken to investigate the solid phase speciation and distribution of americium, neptunium and plutonium which had been experimentally loaded onto different marine sediment types. The chemical leaches employed showed rather poor selectivity but certain trends were evident. Adsorption was not by ion exchange. Americium showed a preferential affinity for carbonate and plutonium for organic matter. Neptunium appeared to have no preferential affinities. Americium was sorbed by acetic acid residues (CaCO 3 removed) and by unleached carbonate-rich sediments with equal efficiency. This indicates that it is able to diversify its solid phase affinity/distribution depending upon which solid phases are available. (author)

  8. Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

    Science.gov (United States)

    Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita; Chaudhary, Vijay K

    2018-01-01

    The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

  9. APPLICATION OF IMMUNOGLOBULIN-BINDING PROTEINS A, G, L IN THE AFFINITY CHROMATOGRAPHY

    Directory of Open Access Journals (Sweden)

    О. V. Sviatenko

    2014-04-01

    Full Text Available Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are used in biotechnology for affinity sorbents production. Сomparative characteristics of proteins A, G, L and affinity sorbents on the basis of them, advantages and disadvantages of these proteins application as ligands in the affinity chromatography are done. Analysis of proteins A, G, L properties is presented. Binding specificities and affinities of these proteins differ between species and antibody subclass. Protein А has high affinity to human IgG1, IgG2, IgG4, mouse IgG2a, IgG2b, IgG3, goat and sheep IgG2, dog, cat, guinea pig, rabbit IgG. Protein G binds strongly to human, mouse, cow, goat, sheep and rabbit IgG. Protein L has ability of strong binding to immunoglobulin kappa-chains of human, mouse, rat and pig. Expediency of application of affinity chromatography with usage of sorbents on the basis of immobilized proteins A, G, L are shown for isolation and purification of antibodies different classes. Previously mentioned method is used as an alternative to conventional methods of protein purification, such as ion-exchange, hydrophobic interactions, metal affinity chromatography, ethanol precipitation due to simplicity in usage, possibility of one-step purification process, obtaining of proteins high level purity, multiuse at maintenance of proper storage and usage conditions. Affinity sorbents on the basis of immobilized proteins A, G, L are used not only for antibodies purification, but also for extraction of different antibodies fractions from blood serum.

  10. Nickel films: Nonselective and selective photochemical deposition and properties

    International Nuclear Information System (INIS)

    Smirnova, N.V.; Boitsova, T.B.; Gorbunova, V.V.; Alekseeva, L.V.; Pronin, V.P.; Kon'uhov, G.S.

    2006-01-01

    Nickel films deposited on quartz surfaces by the photochemical reduction of a chemical nickel plating solution were studied. It was found that the deposition of the films occurs after an induction period, the length of which depends on the composition of the photolyte and the light intensity. Ni particles with a mean diameter of 20-30 nm were detected initially by transmission electron microscopy. The particles then increased in size (50 nm) upon irradiation and grouped into rings consisting of 4-5 particles. Irradiation with high-intensity light produces three-dimensional films. The calculated extinction coefficient of the nickel film was found to be 4800 L mol -1 cm -1 . Electron diffraction revealed that the prepared amorphous nickel films crystallize after one day of storage. It was determined that the films exhibit catalytic activity in the process of nickel deposition from nickel plating solution. The catalytic action remains for about 5-7 min after exposure of the films to air. The processes of selective and nonselective deposition of the nickel films are discussed. The use of poly(butoxy titanium) in the process of selective photochemical deposition enables negative and positive images to be prepared on quartz surfaces

  11. Plasma protein haptoglobin modulates renal iron loading

    DEFF Research Database (Denmark)

    Fagoonee, Sharmila; Gburek, Jakub; Hirsch, Emilio

    2005-01-01

    Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. The strength of hemoglobin binding and the existence of a specific receptor for the haptoglobin-hemoglobin complex in the monocyte/macrophage system clearly suggest that haptoglobin may have a crucial role in heme...... distribution of hemoglobin in haptoglobin-deficient mice resulted in abnormal iron deposits in proximal tubules during aging. Moreover, iron also accumulated in proximal tubules after renal ischemia-reperfusion injury or after an acute plasma heme-protein overload caused by muscle injury, without affecting...... morphological and functional parameters of renal damage. These data demonstrate that haptoglobin crucially prevents glomerular filtration of hemoglobin and, consequently, renal iron loading during aging and following acute plasma heme-protein overload....

  12. Loading direction regulates the affinity of ADP for kinesin.

    Science.gov (United States)

    Uemura, Sotaro; Ishiwata, Shin'ichi

    2003-04-01

    Kinesin is an ATP-driven molecular motor that moves processively along a microtubule. Processivity has been explained as a mechanism that involves alternating single- and double-headed binding of kinesin to microtubules coupled to the ATPase cycle of the motor. The internal load imposed between the two bound heads has been proposed to be a key factor regulating the ATPase cycle in each head. Here we show that external load imposed along the direction of motility on a single kinesin molecule enhances the binding affinity of ADP for kinesin, whereas an external load imposed against the direction of motility decreases it. This coupling between loading direction and enzymatic activity is in accord with the idea that the internal load plays a key role in the unidirectional and cooperative movement of processive motors.

  13. Shear Stress in Nickel and Ni-60Co under One-Dimensional Shock Loading

    International Nuclear Information System (INIS)

    Workman, A.; Wallwork, A.; Meziere, Y. J. E.; Millett, J. C. F.; Bourne, N. K.

    2006-01-01

    The dynamic response of pure nickel (Ni), and its alloy, Ni-60Co (by weight %), has been investigated during one-dimensional shock loading. Few materials' properties are different and the only significantly altered feature is the reduced stacking fault energy (SFE) for the Ni-60Co. This paper considers the effect of this reduced SFE on the shear strength. Data (in terms of shock stress, particle velocity and shock velocity) are also presented. The influence on the shear stress, τ of cobalt additions in nickel are then investigated and presented. Results indicate that the lateral stress is increasing in both materials with the increasing impact stress. The shear stress was found to be higher in the nickel than in the Ni-60Co. The progressive decrease of the lateral stress noted during loading indicates a complex mechanism of deformation behind the shock front

  14. Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.

    Science.gov (United States)

    Holstein, Carly A; Chevalier, Aaron; Bennett, Steven; Anderson, Caitlin E; Keniston, Karen; Olsen, Cathryn; Li, Bing; Bales, Brian; Moore, David R; Fu, Elain; Baker, David; Yager, Paul

    2016-02-01

    To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

  15. Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins.

    Science.gov (United States)

    Pina, Ana Sofia; Carvalho, Sara; Dias, Ana Margarida G C; Guilherme, Márcia; Pereira, Alice S; Caraça, Luciana T; Coroadinha, Ana Sofia; Lowe, Christopher R; Roque, A Cecília A

    2016-11-11

    A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml -1 and 0.48mgml -1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 10 6 M -1 affinity constants and Qmax values of 19.11±2.60ugg -1 and 79.39ugg -1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Use and optimization of a dual-flowrate loading strategy to maximize throughput in protein-a affinity chromatography.

    Science.gov (United States)

    Ghose, Sanchayita; Nagrath, Deepak; Hubbard, Brian; Brooks, Clayton; Cramer, Steven M

    2004-01-01

    The effect of an alternate strategy employing two different flowrates during loading was explored as a means of increasing system productivity in Protein-A chromatography. The effect of such a loading strategy was evaluated using a chromatographic model that was able to accurately predict experimental breakthrough curves for this Protein-A system. A gradient-based optimization routine is carried out to establish the optimal loading conditions (initial and final flowrates and switching time). The two-step loading strategy (using a higher flowrate during the initial stages followed by a lower flowrate) was evaluated for an Fc-fusion protein and was found to result in significant improvements in process throughput. In an extension of this optimization routine, dynamic loading capacity and productivity were simultaneously optimized using a weighted objective function, and this result was compared to that obtained with the single flowrate. Again, the dual-flowrate strategy was found to be superior.

  17. Affinity purification combined with mass spectrometry to identify herpes simplex virus protein-protein interactions.

    Science.gov (United States)

    Meckes, David G

    2014-01-01

    The identification and characterization of herpes simplex virus protein interaction complexes are fundamental to understanding the molecular mechanisms governing the replication and pathogenesis of the virus. Recent advances in affinity-based methods, mass spectrometry configurations, and bioinformatics tools have greatly increased the quantity and quality of protein-protein interaction datasets. In this chapter, detailed and reliable methods that can easily be implemented are presented for the identification of protein-protein interactions using cryogenic cell lysis, affinity purification, trypsin digestion, and mass spectrometry.

  18. Toxic effects of lead and nickel nitrate on rat liver chromatin components.

    Science.gov (United States)

    Rabbani-Chadegani Iii, Azra; Fani, Nesa; Abdossamadi, Sayeh; Shahmir, Nosrat

    2011-01-01

    The biological activity of heavy metals is related to their physicochemical interaction with biological receptors. In the present study, the effect of low concentrations of nickel nitrate and lead nitrate (lead nitrate to chromatin compared to nickel nitrate. Also, the binding affinity of lead nitrate to histone proteins free in solution was higher than nickel. On the basis of the results, it is concluded that lead reacts with chromatin components even at very low concentrations and induce chromatin aggregation through histone-DNA cross-links. Whereas, nickel nitrate is less effective on chromatin at low concentrations, suggesting higher toxicity of lead nitrate on chromatin compared to nickel. Copyright © 2010 Wiley Periodicals, Inc.

  19. Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins

    Science.gov (United States)

    Kaur, Jasvir; Reinhardt, Dieter P.

    2012-01-01

    Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls. PMID:23119075

  20. Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes

    Energy Technology Data Exchange (ETDEWEB)

    Martin-Diaconescu, Vlad; Joseph, Crisjoe A.; Boer, Jodi L.; Mulrooney, Scott B.; Hausinger, Robert P.; Maroney, Michael J.

    2016-12-21

    Nickel-dependent ureases are activated by a multiprotein complex that includes the GTPase UreG. Prior studies showed that nucleotide-free UreG from Klebsiella aerogenes is monomeric and binds one nickel or zinc ion with near-equivalent affinity using an undefined binding site, whereas nucleotide-free UreG from Helicobacter pylori selectively binds one zinc ion per dimer via a universally conserved Cys-Pro-His motif in each protomer. Iodoacetamide-treated K. aerogenes UreG was nearly unaffected in nickel binding compared to non-treated sample, suggesting the absence of thiolate ligands to the metal. X-ray absorption spectroscopy of nickel-bound UreG showed the metal possessed four-coordinate geometry with all O/N donor ligands including one imidazole, thus confirming the absence of thiolate ligation. The nickel site in Strep-tag II-modified protein possessed six-coordinate geometry, again with all O/N donor ligands, but now including two or three imidazoles. An identical site was noted for the Strep-tag II-modified H74A variant, substituted in the Cys-Pro-His motif, ruling out coordination by this His residue. These results are consistent with metal binding to both His6 and a His residue of the fusion peptide in Strep-tagged K. aerogenes UreG. We conclude that the nickel- and zinc-binding site in nucleotide-free K. aerogenes UreG is distinct from that of nucleotide-free H. pylori UreG and does not involve the Cys-Pro-His motif. Further, we show the Strep-tag II can perturb metal coordination of this protein.

  1. Protein selectivity with immobilized metal ion-tacn sorbents: chromatographic studies with human serum proteins and several other globular proteins.

    Science.gov (United States)

    Jiang, W; Graham, B; Spiccia, L; Hearn, M T

    1998-01-01

    The chromatographic selectivity of the immobilized chelate system, 1,4,7-triazocyclononane (tacn), complexed with the borderline metal ions Cu2+, Cr3+, Mn2+, Co2+, Zn2+, and Ni2+ has been investigated with hen egg white lysozyme, horse heart cytochrome c, and horse skeletal muscle myoglobin, as well as proteins present in partially fractionated preparations of human plasma. The effects of ionic strength and pH of the loading and elution buffers on protein selectivities of these new immobilized metal ion affinity chromatographic (IMAC) systems have been examined. The results confirm that immobilized Mn;pl-tacn sorbents exhibit a novel type of IMAC behavior with proteins. In particular, the chromatographic properties of these immobilized M(n+)-tacn ligand systems were significantly different compared to the IMAC behavior observed with other types of immobilized tri- and tetradentate chelating ligands, such as iminodiacetic acid, O-phosphoserine, or nitrilotriacetic acid, when complexed with borderline metal ions. The experimental results have consequently been evaluated in terms of the additional contributions to the interactive processes mediated by effects other than solely the conventional lone pair Lewis soft acid-Lewis soft base coordination interactions, typically found for the IMAC of proteins with borderline and soft metal ions, such as Cu2+ or Ni2+.

  2. Selecting for Fast Protein-Protein Association As Demonstrated on a Random TEM1 Yeast Library Binding BLIP.

    Science.gov (United States)

    Cohen-Khait, Ruth; Schreiber, Gideon

    2018-04-27

    Protein-protein interactions mediate the vast majority of cellular processes. Though protein interactions obey basic chemical principles also within the cell, the in vivo physiological environment may not allow for equilibrium to be reached. Thus, in vitro measured thermodynamic affinity may not provide a complete picture of protein interactions in the biological context. Binding kinetics composed of the association and dissociation rate constants are relevant and important in the cell. Therefore, changes in protein-protein interaction kinetics have a significant impact on the in vivo activity of the proteins. The common protocol for the selection of tighter binders from a mutant library selects for protein complexes with slower dissociation rate constants. Here we describe a method to specifically select for variants with faster association rate constants by using pre-equilibrium selection, starting from a large random library. Toward this end, we refine the selection conditions of a TEM1-β-lactamase library against its natural nanomolar affinity binder β-lactamase inhibitor protein (BLIP). The optimal selection conditions depend on the ligand concentration and on the incubation time. In addition, we show that a second sort of the library helps to separate signal from noise, resulting in a higher percent of faster binders in the selected library. Fast associating protein variants are of particular interest for drug development and other biotechnological applications.

  3. Towards understanding of Nipah virus attachment protein assembly and the role of protein affinity and crowding for membrane curvature events.

    Energy Technology Data Exchange (ETDEWEB)

    Stachowiak, Jeanne C.; Hayden, Carl C.; Negrete, Oscar.; Davis, Ryan Wesley; Sasaki, Darryl Y

    2013-10-01

    Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using point mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.

  4. Extreme disorder in an ultrahigh-affinity protein complex

    DEFF Research Database (Denmark)

    Borgia, Alessandro; Borgia, Madeleine B; Bugge, Katrine

    2018-01-01

    Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions...... with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring...... or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex...

  5. Affinity resins as new tools for identifying target proteins of ascorbic acid.

    Science.gov (United States)

    Iwaoka, Yuji; Nishino, Kohei; Ishikawa, Takahiro; Ito, Hideyuki; Sawa, Yoshihiro; Tai, Akihiro

    2018-02-12

    l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

  6. Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation

    Directory of Open Access Journals (Sweden)

    Zarei Saeed

    2011-08-01

    Full Text Available Abstract Background Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in Escherichia coli and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices. Results Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel did not work well with respect to specific binding capacity and recovery profile. Conclusions Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also

  7. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    Science.gov (United States)

    Salehi, Nasrin; Peng, Ching-An

    2016-07-08

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

  8. Optimizing scoring function of protein-nucleic acid interactions with both affinity and specificity.

    Directory of Open Access Journals (Sweden)

    Zhiqiang Yan

    Full Text Available Protein-nucleic acid (protein-DNA and protein-RNA recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions.

  9. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  10. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    International Nuclear Information System (INIS)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-01-01

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K d 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K d 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy

  11. Robotic high-throughput purification of affinity-tagged recombinant proteins.

    Science.gov (United States)

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories.

  12. Extreme disorder in an ultrahigh-affinity protein complex

    Science.gov (United States)

    Borgia, Alessandro; Borgia, Madeleine B.; Bugge, Katrine; Kissling, Vera M.; Heidarsson, Pétur O.; Fernandes, Catarina B.; Sottini, Andrea; Soranno, Andrea; Buholzer, Karin J.; Nettels, Daniel; Kragelund, Birthe B.; Best, Robert B.; Schuler, Benjamin

    2018-03-01

    Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

  13. Insights into structural features determining odorant affinities to honey bee odorant binding protein 14.

    Science.gov (United States)

    Schwaighofer, Andreas; Pechlaner, Maria; Oostenbrink, Chris; Kotlowski, Caroline; Araman, Can; Mastrogiacomo, Rosa; Pelosi, Paolo; Knoll, Wolfgang; Nowak, Christoph; Larisika, Melanie

    2014-04-18

    Molecular interactions between odorants and odorant binding proteins (OBPs) are of major importance for understanding the principles of selectivity of OBPs towards the wide range of semiochemicals. It is largely unknown on a structural basis, how an OBP binds and discriminates between odorant molecules. Here we examine this aspect in greater detail by comparing the C-minus OBP14 of the honey bee (Apis mellifera L.) to a mutant form of the protein that comprises the third disulfide bond lacking in C-minus OBPs. Affinities of structurally analogous odorants featuring an aromatic phenol group with different side chains were assessed based on changes of the thermal stability of the protein upon odorant binding monitored by circular dichroism spectroscopy. Our results indicate a tendency that odorants show higher affinity to the wild-type OBP suggesting that the introduced rigidity in the mutant protein has a negative effect on odorant binding. Furthermore, we show that OBP14 stability is very sensitive to the position and type of functional groups in the odorant. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

    Science.gov (United States)

    Murphy, Patrick J. M.; Stone, Orrin J.; Anderson, Michelle E.

    2011-01-01

    In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media

  15. Simulating the influence of plasma protein on measured receptor affinity in biochemical assays reveals the utility of Schild analysis for estimating compound affinity for plasma proteins.

    Science.gov (United States)

    Blakeley, D; Sykes, D A; Ensor, P; Bertran, E; Aston, P J; Charlton, S J

    2015-11-01

    Plasma protein binding (PPB) influences the free fraction of drug available to bind to its target and is therefore an important consideration in drug discovery. While traditional methods for assessing PPB (e.g. rapid equilibrium dialysis) are suitable for comparing compounds with relatively weak PPB, they are not able to accurately discriminate between highly bound compounds (typically >99.5%). The aim of the present work was to use mathematical modelling to explore the potential utility of receptor binding and cellular functional assays to estimate the affinity of compounds for plasma proteins. Plasma proteins are routinely added to in vitro assays, so a secondary goal was to investigate the effect of plasma proteins on observed ligand-receptor interactions. Using the principle of conservation of mass and the law of mass action, a cubic equation was derived describing the ligand-receptor complex [LR] in the presence of plasma protein at equilibrium. The model demonstrates the profound influence of PPB on in vitro assays and identifies the utility of Schild analysis, which is usually applied to determine receptor-antagonist affinities, for calculating affinity at plasma proteins (termed KP ). We have also extended this analysis to functional effects using operational modelling and demonstrate that these approaches can also be applied to cell-based assay systems. These mathematical models can potentially be used in conjunction with experimental data to estimate drug-plasma protein affinities in the earliest phases of drug discovery programmes. © 2015 The British Pharmacological Society.

  16. Mua (HP0868) Is a Nickel-Binding Protein That Modulates Urease Activity in Helicobacter pylori

    Science.gov (United States)

    Benoit, Stéphane L.; Maier, Robert J.

    2011-01-01

    A novel mechanism aimed at controlling urease expression in Helicobacter pylori in the presence of ample nickel is described. Higher urease activities were observed in an hp0868 mutant (than in the wild type) in cells supplemented with nickel, suggesting that the HP0868 protein (herein named Mua for modulator of urease activity) represses urease activity when nickel concentrations are ample. The increase in urease activity in the Δmua mutant was linked to an increase in urease transcription and synthesis, as shown by quantitative real-time PCR, SDS-PAGE, and immunoblotting against UreAB. Increased urease synthesis was also detected in a Δmua ΔnikR double mutant strain. The Δmua mutant was more sensitive to nickel toxicity but more resistant to acid challenge than was the wild-type strain. Pure Mua protein binds 2 moles of Ni2+ per mole of dimer. Electrophoretic mobility shift assays did not reveal any binding of Mua to the ureA promoter or other selected promoters (nikR, arsRS, 5′ ureB-sRNAp). Previous yeast two-hybrid studies indicated that Mua and RpoD may interact; however, only a weak interaction was detected via cross-linking with pure components and this could not be verified by another approach. There was no significant difference in the intracellular nickel level between wild-type and mua mutant cells. Taken together, our results suggest the HP0868 gene product represses urease transcription when nickel levels are high through an as-yet-uncharacterized mechanism, thus counterbalancing the well-described NikR-mediated activation. PMID:21505055

  17. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  18. N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

    Science.gov (United States)

    Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

    2015-07-01

    The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.

  19. Load Deflection Characteristics of Nickel Titanium Initial Archwires

    Directory of Open Access Journals (Sweden)

    Hossein Aghili

    2016-05-01

    Full Text Available Objectives: The aim of this study was to assess and compare the characteristics of commonly used initial archwires by their load deflection graphs.Materials and Methods: This study tested three wire designs namely copper nickel titanium (CNT, nickel titanium (NiTi, and multi-strand NiTi (MSNT archwires engaged in passive self-ligating (PSL brackets, active self-ligating (ASL brackets or conventional brackets. To evaluate the mechanical characteristics of the specimens, a three-point bending test was performed. The testing machine vertically applied force on the midpoint of the wire between the central incisor and canine teeth to obtain 2 and 4mm of deflection. The force level at maximum deflection and characteristics of plateau (the average plateau load and the plateau length were recorded. Two-way ANOVA and Tukey’s test were used at P <0.05 level of significance.Results: Force level at maximum deflection and plateau length were significantly affected by the amount of deflection. The type of archwires and brackets had significant effects on force level at maximum deflection, and plateau length. However, the bracket type had no significant effect on the average plateau force.Conclusion: With any type of brackets in deflections of 2 and 4mm, MSNT wire exerted the lowest while NiTi wire exerted the highest force level at maximum deflection and plateau phase. The force level at maximum deflection and the plateau length increased with raising the amount of primary deflection; however the average plateau force did not change significantly.

  20. The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs

    Science.gov (United States)

    Giannone, Richard J.; Liu, Yie; Wang, Yisong

    Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

  1. The release of nickel from orthodontic NiTi wires is increased by dynamic mechanical loading but not constrained by surface nitridation.

    Science.gov (United States)

    Peitsch, T; Klocke, A; Kahl-Nieke, B; Prymak, O; Epple, M

    2007-09-01

    The influence of dynamic mechanical loading and of surface nitridation on the nickel release from superelastic nickel-titanium orthodontic wires was investigated under ultrapure conditions. Commercially available superelastic NiTi arch wires (size 0.018 x 0.025'') without surface modification (Neo Sentalloy) and with nitrogen ion implantation surface treatment (Neo Sentalloy Ionguard) were analyzed. Mechanical loading of wire segments with a force similar to the physiological situation was performed with a frequency of 5 Hz in ultrapure water and saline solution, respectively. The release of nickel was monitored by atomic absorption spectroscopy for up to 36 days. The mechanically loaded wires released significantly more nickel ( approximately 45 ng cm(-2) d(-1)) than did nonloaded wires (<1 ng cm(-2) d(-1)). There was no statistically significant effect of the testing solution (water or NaCl) or of the surface nitridation. The total amount of released nickel was small in all cases, but may nevertheless account for the occasional clinical observations of adverse reactions during application of NiTi-based orthodontic appliances. The surface nitridation did not constrain the release of nickel from NiTi under continuous mechanical stress.

  2. Semi-continuous protein fractionating using affinity cross-flow filtration

    NARCIS (Netherlands)

    Borneman, Zandrie; Zhang, W.; van den Boomgaard, Anthonie; Smolders, C.A.

    2002-01-01

    Protein purification by means of downstream processing is increasingly important. At the University of Twente a semi-continuous process is developed for the isolation of BSA out of crude protein mixtures. For this purpose an automated Affinity Cross-Flow Filtration, ACFF, process is developed. This

  3. Modeling a constant power load for nickel-hydrogen battery testing using SPICE

    Science.gov (United States)

    Bearden, Douglas B.; Lollar, Louis F.; Nelms, R. M.

    1990-01-01

    The effort to design and model a constant power load for the HST (Hubble Space Telescope) nickel-hydrogen battery tests is described. The constant power load was designed for three different simulations on the batteries: life cycling, reconditioning, and capacity testing. A dc-dc boost converter was designed to act as this constant power load. A boost converter design was chosen because of the low test battery voltage (4 to 6 VDC) generated and the relatively high power requirement of 60 to 70 W. The SPICE model was shown to consistently predict variations in the actual circuit as various designs were attempted. It is concluded that the confidence established in the SPICE model of the constant power load ensures its extensive utilization in future efforts to improve performance in the actual load circuit.

  4. Expression and affinity purification of recombinant proteins from plants

    Science.gov (United States)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  5. Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

    International Nuclear Information System (INIS)

    Choi, Ryan; Kelley, Angela; Leibly, David; Nakazawa Hewitt, Stephen; Napuli, Alberto; Van Voorhis, Wesley

    2011-01-01

    An overview of the methods used for high-throughput cloning and protein-expression screening of SSGCID hexahistidine recombinant proteins is provided. It is demonstrated that screening for recombinant proteins that are highly recoverable from immobilized metal-affinity chromatography improves the likelihood that a protein will produce a structure. The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure

  6. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brian H. Carrick

    2016-03-01

    Full Text Available Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  7. Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine

    International Nuclear Information System (INIS)

    Koehrle, J.R.; Rasmussen, U.B.; Rokos, H.; Leonard, J.L.; Hesch, R.D.

    1990-01-01

    125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney

  8. Selective recovery of nickel over iron from a nickel-iron solution using microbial sulfate reduction in a gas-lift bioreactor

    NARCIS (Netherlands)

    Bijmans, M.F.M.; Helvoort, van P.J.; Dar, S.; Dopson, M.; Lens, P.N.L.; Buisman, C.J.N.

    2009-01-01

    Process streams with high concentrations of metals and sulfate are characteristic for the mining and metallurgical industries. This study aims to selectively recover nickel from a nickel-iron-containing solution at pH 5.0 using a single stage bioreactor that simultaneously combines low pH sulfate

  9. Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

    Science.gov (United States)

    Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

    2017-11-01

    Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  10. Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition.

    Directory of Open Access Journals (Sweden)

    Yves Nominé

    Full Text Available Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15 derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F was equally well tolerated as the wild type glutamine (Q at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv-peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology. Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes.

  11. The development of an affinity evaluation and prediction system by using protein–protein docking simulations and parameter tuning

    Directory of Open Access Journals (Sweden)

    Koki Tsukamoto

    2009-01-01

    Full Text Available Koki Tsukamoto1, Tatsuya Yoshikawa1,2, Kiyonobu Yokota1, Yuichiro Hourai1, Kazuhiko Fukui11Computational Biology Research Center (CBRC, National Institute of Advanced Industrial Science and Technology (AIST, Koto-ku, Tokyo, Japan; 2Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Toyonaka, Osaka, JapanAbstract: A system was developed to evaluate and predict the interaction between protein pairs by using the widely used shape complementarity search method as the algorithm for docking simulations between the proteins. We used this system, which we call the affinity evaluation and prediction (AEP system, to evaluate the interaction between 20 protein pairs. The system first executes a “round robin” shape complementarity search of the target protein group, and evaluates the interaction between the complex structures obtained by the search. These complex structures are selected by using a statistical procedure that we developed called ‘grouping’. At a prevalence of 5.0%, our AEP system predicted protein–protein interactions with a 50.0% recall, 55.6% precision, 95.5% accuracy, and an F-measure of 0.526. By optimizing the grouping process, our AEP system successfully predicted 10 protein pairs (among 20 pairs that were biologically relevant combinations. Our ultimate goal is to construct an affinity database that will provide cell biologists and drug designers with crucial information obtained using our AEP system.Keywords: protein–protein interaction, affinity analysis, protein–protein docking, FFT, massive parallel computing

  12. Nickel extraction from nickel matte

    Science.gov (United States)

    Subagja, R.

    2018-01-01

    In present work, the results of research activities to make nickel metal from nickel matte are presented. The research activities were covering a) nickel matte characterization using Inductively Couple plasma (ICP), Electron Probe Micro Analyzer (EPMA) and X-Ray Diffraction (XRD), b) nickel matte dissolution process to dissolve nickel from nickel matte into the spent electrolyte solutions that contains hydrochloric acid, c) purification of nickel chloride leach solution by copper cementation process to remove copper using nickel matte, selective precipitation process to remove iron, solvent extraction using Tri normal octyl amine to separate cobalt from nickel chloride solutions and d) Nickel electro winning process to precipitate nickel into the cathode surface from purified nickel chloride solution by using direct current. The research activities created 99, 72 % pure nickel metal as the final product of the process.

  13. Selective chromogenic detection of thiol-containing biomolecules using carbonaceous nanospheres loaded with silver nanoparticles as carrier.

    Science.gov (United States)

    Hu, Bo; Zhao, Yang; Zhu, Hai-Zhou; Yu, Shu-Hong

    2011-04-26

    Thiol-containing biomolecules show strong affinity with noble metal nanostructures and could not only stably protect them but also control the self-assembly process of these special nanostructures. A highly selective and sensitive chromogenic detection method has been designed for the low and high molecular weight thiol-containing biomolecules, including cysteine, glutathione, dithiothreitol, and bovine serum albumin, using a new type of carbonaceous nanospheres loaded with silver nanoparticles (Ag NPs) as carrier. This strategy relies upon the place-exchange process between the reporter dyes on the surface of Ag NPs and the thiol groups of thiol-containing biomolecules. The concentration of biomolecules can be determined by monitoring with the fluorescence intensity of reporter dyes dispersed in solution. This new chromogenic assay method could selectively detect these biomolecules in the presence of various other amino acids and monosaccharides and even sensitively detect the thiol-containing biomolecules with different molecular weight, even including proteins.

  14. A dual protease approach for expression and affinity purification of recombinant proteins.

    Science.gov (United States)

    Raran-Kurussi, Sreejith; Waugh, David S

    2016-07-01

    We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

  15. Histidine protects against zinc and nickel toxicity in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    John T Murphy

    2011-03-01

    Full Text Available Zinc is an essential trace element involved in a wide range of biological processes and human diseases. Zinc excess is deleterious, and animals require mechanisms to protect against zinc toxicity. To identify genes that modulate zinc tolerance, we performed a forward genetic screen for Caenorhabditis elegans mutants that were resistant to zinc toxicity. Here we demonstrate that mutations of the C. elegans histidine ammonia lyase (haly-1 gene promote zinc tolerance. C. elegans haly-1 encodes a protein that is homologous to vertebrate HAL, an enzyme that converts histidine to urocanic acid. haly-1 mutant animals displayed elevated levels of histidine, indicating that C. elegans HALY-1 protein is an enzyme involved in histidine catabolism. These results suggest the model that elevated histidine chelates zinc and thereby reduces zinc toxicity. Supporting this hypothesis, we demonstrated that dietary histidine promotes zinc tolerance. Nickel is another metal that binds histidine with high affinity. We demonstrated that haly-1 mutant animals are resistant to nickel toxicity and dietary histidine promotes nickel tolerance in wild-type animals. These studies identify a novel role for haly-1 and histidine in zinc metabolism and may be relevant for other animals.

  16. The competitor-introduced Ggamma recruitment system, a new approach for screening affinity-enhanced proteins.

    Science.gov (United States)

    Fukuda, Nobuo; Ishii, Jun; Tanaka, Tsutomu; Kondo, Akihiko

    2010-04-01

    We have developed a new approach based on the Ggamma recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established Ggamma recruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that membrane localization of the G-protein gamma subunit (Ggamma) is essential for signal transduction in yeast. In the original Y2H system, an engineered Ggamma that lacks membrane localization upon deletion of the lipid modification site (Ggamma(cyto)) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with Ggamma(cyto) are expressed. As protein-protein interactions bring Ggamma(cyto) towards the plasma membrane, G-protein signaling can be activated, and the interaction is detected by various cellular responses as the readout. In the current study, we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counterpart-Ggamma(cyto) fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z domain derived from Staphylococcus aureus protein A as candidate proteins or competitors, and the Fc portion of human immunoglobulin G (IgG) as the counterpart, we demonstrate that affinity-enhanced proteins can be effectively screened from a library containing a 10 000-fold excess of non-enhanced proteins. This new approach, called the competitor-introduced Ggamma recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones.

  17. A community standard format for the representation of protein affinity reagents

    DEFF Research Database (Denmark)

    Gloriam, David Erik Immanuel; Orchard, Sandra; Bertinetti, Daniela

    2010-01-01

    Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often...... that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange....... Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of Proteome...

  18. Selective Electroless Nickel Plating on PMMA using Chloroform Pre-Treatment

    Science.gov (United States)

    Sipes, Nicholas

    In the past 5 years, we have discovered that chloroform promotes the adhesion of thin gold films to Poly(methyl methacrylate) surfaces. Based on this new understanding of the interaction of chloroform with PMMA and metal atoms, we were curious to see if chloroform would promote the adhesion of Nickel to PMMA deposited by electroless plating. My goal was to selectively electroless plate Nickel onto PMMA. Chloroform was spun-cast onto 1 inch square PMMA substrates. I used electrical tape to shield one half of the PMMA from the chloroform during spin-casting; this allowed for a direct comparison of treated vs. untreated. The samples were then put through hydrochloric acid and a series of baths provided by Transene Company Inc. to electrolessly deposit nickel on the sample. After many trials, there was a clear distinction in the adhesion strength of the Nickel to the plain PMMA surface vs. the chloroform pre-treated surface. Showing that it is possible to create chloroform sites via spin-casting for electroless nickel plating on PMMA opens up the challenge to better understand the chemistry taking place and to perfect the electroless plating process.

  19. Site-selective electroless nickel plating on patterned thin films of macromolecular metal complexes.

    Science.gov (United States)

    Kimura, Mutsumi; Yamagiwa, Hiroki; Asakawa, Daisuke; Noguchi, Makoto; Kurashina, Tadashi; Fukawa, Tadashi; Shirai, Hirofusa

    2010-12-01

    We demonstrate a simple route to depositing nickel layer patterns using photocross-linked polymer thin films containing palladium catalysts, which can be used as adhesive interlayers for fabrication of nickel patterns on glass and plastic substrates. Electroless nickel patterns can be obtained in three steps: (i) the pattern formation of partially quaterized poly(vinyl pyridine) by UV irradiation, (ii) the formation of macromolecular metal complex with palladium, and (iii) the nickel metallization using electroless plating bath. Metallization is site-selective and allows for a high resolution. And the resulting nickel layered structure shows good adhesion with glass and plastic substrates. The direct patterning of metallic layers onto insulating substrates indicates a great potential for fabricating micro/nano devices.

  20. Long life nickel electrodes for a nickel-hydrogen cell: Cycle life tests

    Science.gov (United States)

    Lim, H. S.; Verzwyvelt, S. A.

    1985-01-01

    In order to develop a long life nickel electrode for a Ni/H2 cell, the cycle life of nickel electrodes was tested in Ni/H2 boiler plate cells. A 19 test cell matrix was made of various nickel electrode designs including three levels each of plaque mechanical strength, median pore size of the plaque, and active material loading. Test cells were cycled to the end of their life (0.5v) in a 45 minute low Earth orbit cycle regime at 80% depth-of-discharge. It is shown that the active material loading level affects the cycle life the most with the optimum loading at 1.6 g/cc void. Mechanical strength does not affect the cycle life noticeably in the bend strength range of 400 to 700 psi. It is found that the best plaque is made of INCO nickel powder type 287 and has median pore size of 13 micron.

  1. Protein-observed (19)F-NMR for fragment screening, affinity quantification and druggability assessment.

    Science.gov (United States)

    Gee, Clifford T; Arntson, Keith E; Urick, Andrew K; Mishra, Neeraj K; Hawk, Laura M L; Wisniewski, Andrea J; Pomerantz, William C K

    2016-08-01

    NMR spectroscopy can be used to quantify the binding affinity between proteins and low-complexity molecules, termed 'fragments'; this versatile screening approach allows researchers to assess the druggability of new protein targets. Protein-observed (19)F-NMR (PrOF NMR) using (19)F-labeled amino acids generates relatively simple spectra that are able to provide dynamic structural information toward understanding protein folding and function. Changes in these spectra upon the addition of fragment molecules can be observed and quantified. This protocol describes the sequence-selective labeling of three proteins (the first bromodomains of Brd4 and BrdT, and the KIX domain of the CREB-binding protein) using commercially available fluorinated aromatic amino acids and fluorinated precursors as example applications of the method developed by our research group. Fragment-screening approaches are discussed, as well as Kd determination, ligand-efficiency calculations and druggability assessment, i.e., the ability to target these proteins using small-molecule ligands. Experiment times on the order of a few minutes and the simplicity of the NMR spectra obtained make this approach well-suited to the investigation of small- to medium-sized proteins, as well as the screening of multiple proteins in the same experiment.

  2. Combining metal oxide affinity chromatography (MOAC and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites

    Directory of Open Access Journals (Sweden)

    Weckwerth Wolfram

    2005-11-01

    Full Text Available Abstract Background Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy. Results Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC and selective ion trap mass spectrometry. The complete approach involves (i enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment, (iii identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv identification of phosphorylation sites of these enriched proteins using automatic detection of H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the corresponding MS2-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures. Conclusion A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to

  3. Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity.

    Science.gov (United States)

    Jin, Rongsheng; Rummel, Andreas; Binz, Thomas; Brunger, Axel T

    2006-12-21

    Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng kg(-1), they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. The molecular details of the toxin-cell recognition have been elusive. Here we report the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15 A resolution. On binding, a helix is induced in the luminal domain which binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. Our results provide the basis for the rational development of preventive vaccines or inhibitors against these neurotoxins.

  4. Identification of high-affinity calmodulin-binding proteins in rat liver

    International Nuclear Information System (INIS)

    Hanley, R.M.; Dedman, J.R.; Shenolikar, S.

    1987-01-01

    The Ca 2+ -dependent binding of [ 125 I] calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or acceptor proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised > 80% of the Ca 2+ -dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding subunit of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca 2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver

  5. Molecular basis for the wide range of affinity found in Csr/Rsm protein-RNA recognition.

    Science.gov (United States)

    Duss, Olivier; Michel, Erich; Diarra dit Konté, Nana; Schubert, Mario; Allain, Frédéric H-T

    2014-04-01

    The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, Kd). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein-RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.

  6. Zeolite Nanoparticles for Selective Sorption of Plasma Proteins.

    Science.gov (United States)

    Rahimi, M; Ng, E-P; Bakhtiari, K; Vinciguerra, M; Ali Ahmad, H; Awala, H; Mintova, S; Daghighi, M; Bakhshandeh Rostami, F; de Vries, M; Motazacker, M M; Peppelenbosch, M P; Mahmoudi, M; Rezaee, F

    2015-11-30

    The affinity of zeolite nanoparticles (diameter of 8-12 nm) possessing high surface area and high pore volume towards human plasma proteins has been investigated. The protein composition (corona) of zeolite nanoparticles has been shown to be more dependent on the plasma protein concentrations and the type of zeolites than zeolite nanoparticles concentration. The number of proteins present in the corona of zeolite nanoparticles at 100% plasma (in vivo state) is less than with 10% plasma exposure. This could be due to a competition between the proteins to occupy the corona of the zeolite nanoparticles. Moreover, a high selective adsorption for apolipoprotein C-III (APOC-III) and fibrinogen on the zeolite nanoparticles at high plasma concentration (100%) was observed. While the zeolite nanoparticles exposed to low plasma concentration (10%) exhibited a high selective adsorption for immunoglobulin gamma (i.e. IGHG1, IGHG2 and IGHG4) proteins. The zeolite nanoparticles can potentially be used for selectively capture of APOC-III in order to reduce the activation of lipoprotein lipase inhibition during hypertriglyceridemia treatment. The zeolite nanoparticles can be adapted to hemophilic patients (hemophilia A (F-VIII deficient) and hemophilia B (F-IX deficient)) with a risk of bleeding, and thus might be potentially used in combination with the existing therapy.

  7. The assembly of the plant urease activation complex and the essential role of the urease accessory protein G (UreG) in delivery of nickel to urease.

    Science.gov (United States)

    Myrach, Till; Zhu, Anting; Witte, Claus-Peter

    2017-09-01

    Urease is a ubiquitous nickel metalloenzyme. In plants, its activation requires three urease accessory proteins (UAPs), UreD, UreF, and UreG. In bacteria, the UAPs interact with urease and facilitate activation, which involves the channeling of two nickel ions into the active site. So far this process has not been investigated in eukaryotes. Using affinity pulldowns of Strep-tagged UAPs from Arabidopsis and rice transiently expressed in planta , we demonstrate that a urease-UreD-UreF-UreG complex exists in plants and show its stepwise assembly. UreG is crucial for nickel delivery because UreG-dependent urease activation in vitro was observed only with UreG obtained from nickel-sufficient plants. This activation competence could not be generated in vitro by incubation of UreG with nickel, bicarbonate, and GTP. Compared with their bacterial orthologs, plant UreGs possess an N-terminal extension containing a His- and Asp/Glu-rich hypervariable region followed by a highly conserved sequence comprising two potential H X H metal-binding sites. Complementing the ureG-1 mutant of Arabidopsis with N-terminal deletion variants of UreG demonstrated that the hypervariable region has a minor impact on activation efficiency, whereas the conserved region up to the first H X H motif is highly beneficial and up to the second H X H motif strictly required for activation. We also show that urease reaches its full activity several days after nickel becomes available in the leaves, indicating that urease activation is limited by nickel accessibility in vivo Our data uncover the crucial role of UreG for nickel delivery during eukaryotic urease activation, inciting further investigations of the details of this process. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Identification of Tyrosine Phosphorylated Proteins by SH2 Domain Affinity Purification and Mass Spectrometry.

    Science.gov (United States)

    Buhs, Sophia; Gerull, Helwe; Nollau, Peter

    2017-01-01

    Phosphotyrosine signaling plays a major role in the control of many important biological functions such as cell proliferation and apoptosis. Deciphering of phosphotyrosine-dependent signaling is therefore of great interest paving the way for the understanding of physiological and pathological processes of signal transduction. On the basis of the specific binding of SH2 domains to phosphotyrosine residues, we here present an experimental workflow for affinity purification and subsequent identification of tyrosine phosphorylated proteins by mass spectrometry. In combination with SH2 profiling, a broadly applicable platform for the characterization of phosphotyrosine profiles in cell extracts, our pull down strategy enables researchers by now to identify proteins in signaling cascades which are differentially phosphorylated and selectively recognized by distinct SH2 domains.

  9. Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

    Science.gov (United States)

    Keefe, Andrew J.; Jiang, Shaoyi

    2012-01-01

    Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein-substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity.

  10. G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes.

    Science.gov (United States)

    O'Neill, Patrick R; Karunarathne, W K Ajith; Kalyanaraman, Vani; Silvius, John R; Gautam, N

    2012-12-18

    Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. G-protein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.

  11. Selective synthesis and characterization of sea urchin-like metallic nickel nanocrystals

    International Nuclear Information System (INIS)

    Liu Xiaohe; Liang Xudong; Zhang Ning; Qiu Guanzhou; Yi Ran

    2006-01-01

    Sea urchin-like nanobelt-based and nanorod-based metallic nickel nanocrystals have been selective synthesized via a hydrothermal reduction route in which sodium hydroxide was used as alkaline reagent and aqueous hydrazine (N 2 H 4 .H 2 O) was used as reducing agent. The morphology and structure of final products could be easily controlled by adjust process parameters such as hydrothermal time, reaction temperature and alkaline concentration. Surfactant cetyltrimethylammonium bromide (CTAB) was also important parameter influencing the morphology of the products. The morphology and phase structure of the final products have been investigated by X-ray diffraction, transmission electron microscopy and selected area electron diffraction. The probable formation mechanism of the sea urchin-like metallic nickel nanocrystals was discussed on the basis of the experimental results

  12. Specificity and affinity motifs for Grb2 SH2-ligand interactions

    NARCIS (Netherlands)

    Kessels, Helmut W. H. G.; Ward, Alister C.; Schumacher, Ton N. M.

    2002-01-01

    Protein-protein interactions are often mediated by the recognition of short continuous amino acid stretches on target proteins by specific binding domains. Affinity-based selection strategies have successfully been used to define recognition motifs for a large series of such protein domains.

  13. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    Full Text Available A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

  14. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    Science.gov (United States)

    Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

    2015-01-01

    A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

  15. Cost Function Network-based Design of Protein-Protein Interactions: predicting changes in binding affinity.

    Science.gov (United States)

    Viricel, Clément; de Givry, Simon; Schiex, Thomas; Barbe, Sophie

    2018-02-20

    Accurate and economic methods to predict change in protein binding free energy upon mutation are imperative to accelerate the design of proteins for a wide range of applications. Free energy is defined by enthalpic and entropic contributions. Following the recent progresses of Artificial Intelligence-based algorithms for guaranteed NP-hard energy optimization and partition function computation, it becomes possible to quickly compute minimum energy conformations and to reliably estimate the entropic contribution of side-chains in the change of free energy of large protein interfaces. Using guaranteed Cost Function Network algorithms, Rosetta energy functions and Dunbrack's rotamer library, we developed and assessed EasyE and JayZ, two methods for binding affinity estimation that ignore or include conformational entropic contributions on a large benchmark of binding affinity experimental measures. If both approaches outperform most established tools, we observe that side-chain conformational entropy brings little or no improvement on most systems but becomes crucial in some rare cases. as open-source Python/C ++ code at sourcesup.renater.fr/projects/easy-jayz. thomas.schiex@inra.fr and sophie.barbe@insa-toulouse.fr. Supplementary data are available at Bioinformatics online.

  16. Affinity-based, biophysical methods to detect and analyze ligand binding to recombinant proteins: matching high information content with high throughput.

    Science.gov (United States)

    Holdgate, Geoff A; Anderson, Malcolm; Edfeldt, Fredrik; Geschwindner, Stefan

    2010-10-01

    Affinity-based technologies have become impactful tools to detect, monitor and characterize molecular interactions using recombinant target proteins. This can aid the understanding of biological function by revealing mechanistic details, and even more importantly, enables the identification of new improved ligands that can modulate the biological activity of those targets in a desired fashion. The selection of the appropriate technology is a key step in that process, as each one of the currently available technologies offers a characteristic type of biophysical information about the ligand-binding event. Alongside the indisputable advantages of each of those technologies they naturally display diverse restrictions that are quite frequently related to the target system to be studied but also to the affinity, solubility and molecular size of the ligands. This paper discusses some of the theoretical and experimental aspects of the most common affinity-based methods, what type of information can be gained from each one of those approaches, and what requirements as well as limitations are expected from working with recombinant proteins on those platforms and how those can be optimally addressed.

  17. Selective synthesis and characterization of sea urchin-like metallic nickel nanocrystals

    Energy Technology Data Exchange (ETDEWEB)

    Liu Xiaohe [Department of Inorganic Materials, Central South University, Changsha, Hunan 410083 (China)]. E-mail: liuxh@mail.csu.edu.cn; Liang Xudong [Department of Inorganic Materials, Central South University, Changsha, Hunan 410083 (China); Zhang Ning [Department of Inorganic Materials, Central South University, Changsha, Hunan 410083 (China); Qiu Guanzhou [Department of Inorganic Materials, Central South University, Changsha, Hunan 410083 (China); Yi Ran [Department of Inorganic Materials, Central South University, Changsha, Hunan 410083 (China)

    2006-08-15

    Sea urchin-like nanobelt-based and nanorod-based metallic nickel nanocrystals have been selective synthesized via a hydrothermal reduction route in which sodium hydroxide was used as alkaline reagent and aqueous hydrazine (N{sub 2}H{sub 4}.H{sub 2}O) was used as reducing agent. The morphology and structure of final products could be easily controlled by adjust process parameters such as hydrothermal time, reaction temperature and alkaline concentration. Surfactant cetyltrimethylammonium bromide (CTAB) was also important parameter influencing the morphology of the products. The morphology and phase structure of the final products have been investigated by X-ray diffraction, transmission electron microscopy and selected area electron diffraction. The probable formation mechanism of the sea urchin-like metallic nickel nanocrystals was discussed on the basis of the experimental results.

  18. Nickel electrodes as a cheap and versatile platform for studying structure and function of immobilized redox proteins

    Energy Technology Data Exchange (ETDEWEB)

    Han, Xiao Xia [State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, Changchun 130012 (China); Institut für Chemie, Technische Universität Berlin, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin (Germany); Li, Junbo [State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, Changchun 130012 (China); Öner, Ibrahim Halil [Institut für Chemie, Technische Universität Berlin, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin (Germany); Zhao, Bing [State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, Changchun 130012 (China); Leimkühler, Silke [Institut für Biochemie und Biologie, Universität Potsdam, Karl-Liebknecht Straße 24-25, H. 25, Golm D-14476 (Germany); Hildebrandt, Peter [Institut für Chemie, Technische Universität Berlin, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin (Germany); Weidinger, Inez M., E-mail: i.weidinger@mailbox.tu-berlin.de [Institut für Chemie, Technische Universität Berlin, Sekr. PC14, Strasse des 17. Juni 135, D-10623 Berlin (Germany)

    2016-10-19

    Practical use of many bioelectronic and bioanalytical devices is limited by the need of expensive materials and time consuming fabrication. Here we demonstrate the use of nickel electrodes as a simple and cheap solid support material for bioelectronic applications. The naturally nanostructured electrodes showed a surprisingly high electromagnetic surface enhancement upon light illumination such that immobilization and electron transfer reactions of the model redox proteins cytochrome b{sub 5} (Cyt b{sub 5}) and cytochrome c (Cyt c) could be followed via surface enhanced resonance Raman spectroscopy. It could be shown that the nickel surface, when used as received, promotes a very efficient binding of the proteins upon preservation of their native structure. The immobilized redox proteins could efficiently exchange electrons with the electrode and could even act as an electron relay between the electrode and solubilized myoglobin. Our results open up new possibility for nickel electrodes as an exceptional good support for bioelectronic devices and biosensors on the one hand and for surface enhanced spectroscopic investigations on the other hand. - Highlights: • Nickel electrodes were used without further functionalization as supports for various redox proteins. • It was possible to monitor the immobilized proteins via surface enhanced Raman spectroscopy. • The native structure of the immobilized proteins was preserved and they could exchange electrons with the Ni electrode. • The immobilized redox proteins worked as an electron relay between electrode and solubilized myoglobin.

  19. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    Science.gov (United States)

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.

  20. Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies.

    Science.gov (United States)

    Julian, Mark C; Lee, Christine C; Tiller, Kathryn E; Rabia, Lilia A; Day, Evan K; Schick, Arthur J; Tessier, Peter M

    2015-10-01

    An attractive approach for designing lead antibody candidates is to mimic natural protein interactions by grafting peptide recognition motifs into the complementarity-determining regions (CDRs). We are using this approach to generate single-domain (VH) antibodies specific for amyloid-forming proteins such as the Alzheimer's Aβ peptide. Here, we use random mutagenesis and yeast surface display to improve the binding affinity of a lead VH domain grafted with Aβ residues 33-42 in CDR3. Interestingly, co-selection for improved Aβ binding and VH display on the surface of yeast yields antibody domains with improved affinity and reduced stability. The highest affinity VH domains were strongly destabilized on the surface of yeast as well as unfolded when isolated as autonomous domains. In contrast, stable VH domains with improved affinity were reliably identified using yeast surface display by replacing the display antibody that recognizes a linear epitope tag at the terminus of both folded and unfolded VH domains with a conformational ligand (Protein A) that recognizes a discontinuous epitope on the framework of folded VH domains. Importantly, we find that selection for improved stability using Protein A without simultaneous co-selection for improved Aβ binding leads to strong enrichment for stabilizing mutations that reduce antigen binding. Our findings highlight the importance of simultaneously optimizing affinity and stability to improve the rapid isolation of well-folded and specific antibody fragments. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Biological role of nickel

    Energy Technology Data Exchange (ETDEWEB)

    Thauer, R K; Diekert, G; Schoenheit, P

    1980-01-01

    Several enzymes and one cofactor have recently been shown to contain nickel. For example, urease of jack beans has been found to be a nickel protein and factor F/sub 430/ from methanogenic bacteria to be a nickel tetrapyrrole. The biological role of nickel in several organisms is discussed.

  2. Selective Impairment of Auditory Selective Attention under Concurrent Cognitive Load

    Science.gov (United States)

    Dittrich, Kerstin; Stahl, Christoph

    2012-01-01

    Load theory predicts that concurrent cognitive load impairs selective attention. For visual stimuli, it has been shown that this impairment can be selective: Distraction was specifically increased when the stimulus material used in the cognitive load task matches that of the selective attention task. Here, we report four experiments that…

  3. Pinning of size-selected gold and nickel nanoclusters on graphite

    NARCIS (Netherlands)

    Di Vece, M.|info:eu-repo/dai/nl/248753355; Paloma, S.; Palmer, R.E.

    2005-01-01

    Size-selected gold and nickel nanoclusters are of interest from an electronic, catalytic, and biological point of view. These applications require the deposition of the clusters on a surface, and a key challenge is to retain the cluster size. Here controlled energy impact is used to immobilize the

  4. Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

    Science.gov (United States)

    Oshiro, Satoshi; Honda, Shinya

    2014-04-18

    Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

  5. Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels

    DEFF Research Database (Denmark)

    Domanski, Michal; Molloy, Kelly; Jiang, Hua

    2012-01-01

    An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous...

  6. Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding.

    Science.gov (United States)

    Paramelle, David; Peng, Tao; Free, Paul; Fernig, David G; Lim, Sierin; Tomczak, Nikodem

    2016-01-01

    Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages' pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages' core and low non-specific binding to the cages' outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage's core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently

  7. Selective high-affinity polydentate ligands and methods of making such

    Energy Technology Data Exchange (ETDEWEB)

    Denardo, Sally J.; Denardo, Gerald L.; Balhorn, Rodney L.

    2018-02-06

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  8. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

    Science.gov (United States)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Metal-loaded SBA-16-like silica – Correlation between basicity and affinity towards hydrogen

    International Nuclear Information System (INIS)

    Ouargli-Saker, R.; Bouazizi, N.; Boukoussa, B.; Barrimo, Diana; Paola-Nunes-Beltrao, Ana; Azzouz, A.

    2017-01-01

    Highlights: • Metal dispersion in longitudinal channels confers adsorption properties to SBA-16. • Both Fe"0-NPs and Cu"0-NPs seem to be responsible of this effect. • Effect of the repetitive adsorption-desorption cycles on CO_2 and water sorption. • Hydrogen storage on the functionalized materials. - Abstract: Nanoparticles of Cu"o (CuNPs) and Fe"o (FeNPs) were dispersed in SBA-16-like silica, resulting metal-loaded materials (Cu-SBA-16 and Fe-SBA-16) with improved affinity towards hydrogen. Electron microscopy and X-ray diffraction showed that MNP dispersion occurs mainly inside SBA-16 channels. MNP incorporation was found to confer affinity to the silica surface, since higher CO_2 retention capacity (CRC) was registered Cu/SBA-16 and Fe/SBA-16. This was accompanied by a significant improvement of the affinity towards hydrogen, as supported by hydrogen adsorption tests. This was explained in terms of strong hydrogen interaction with MNP and lattice oxygen atoms. The results reported herein open new prospects for SBA-16 as potential adsorbents for hydrogen storage.

  10. Metal-loaded SBA-16-like silica – Correlation between basicity and affinity towards hydrogen

    Energy Technology Data Exchange (ETDEWEB)

    Ouargli-Saker, R. [Department of Materials Engineering, University of Science and Technology, El M’naouer, BP 1505, Oran (Algeria); Nanoqam, Department of Chemistry, University of Quebec at Montreal, H3C3P8 (Canada); Bouazizi, N. [Nanoqam, Department of Chemistry, University of Quebec at Montreal, H3C3P8 (Canada); Unité de recherche, Electrochimie, Matériaux et Environnement, Faculté des Sciences de Gabès, Université de Gabès, Cité Erriadh, 6072 Gabès (Tunisia); Boukoussa, B. [Department of Materials Engineering, University of Science and Technology, El M’naouer, BP 1505, Oran (Algeria); Lqamb, Laboratório de Química Analítica Ambiental, Faculdade de Química, Pontifícia Universidade Católica do Rio Grande do Sul (Brazil); Barrimo, Diana [Nanoqam, Department of Chemistry, University of Quebec at Montreal, H3C3P8 (Canada); Paola-Nunes-Beltrao, Ana [Nanoqam, Department of Chemistry, University of Quebec at Montreal, H3C3P8 (Canada); Laboratory of Materials Chemistry L.C.M, University of Oran1 Ahmed Ben Bella, BP 1524, El-Mnaouer, 31000 Oran (Algeria); Azzouz, A., E-mail: azzouz.a@uqam.ca [Nanoqam, Department of Chemistry, University of Quebec at Montreal, H3C3P8 (Canada)

    2017-07-31

    Highlights: • Metal dispersion in longitudinal channels confers adsorption properties to SBA-16. • Both Fe{sup 0}-NPs and Cu{sup 0}-NPs seem to be responsible of this effect. • Effect of the repetitive adsorption-desorption cycles on CO{sub 2} and water sorption. • Hydrogen storage on the functionalized materials. - Abstract: Nanoparticles of Cu{sup o} (CuNPs) and Fe{sup o} (FeNPs) were dispersed in SBA-16-like silica, resulting metal-loaded materials (Cu-SBA-16 and Fe-SBA-16) with improved affinity towards hydrogen. Electron microscopy and X-ray diffraction showed that MNP dispersion occurs mainly inside SBA-16 channels. MNP incorporation was found to confer affinity to the silica surface, since higher CO{sub 2} retention capacity (CRC) was registered Cu/SBA-16 and Fe/SBA-16. This was accompanied by a significant improvement of the affinity towards hydrogen, as supported by hydrogen adsorption tests. This was explained in terms of strong hydrogen interaction with MNP and lattice oxygen atoms. The results reported herein open new prospects for SBA-16 as potential adsorbents for hydrogen storage.

  11. Nickel hydrogen/nickel cadmium battery trade studies

    Science.gov (United States)

    Stadnick, S. J.

    1983-01-01

    Nickel Hydrogen cell and battery technology has matured to the point where a real choice exists between Nickel Hydrogen and Nickel Cadmium batteries for each new spacecraft application. During the past few years, a number of spacecraft programs have been evaluated at Hughes with respect to this choice, with the results being split about fifty-fifty. The following paragraphs contain criteria which were used in making the battery selection.

  12. Selected-area growth of nickel micropillars on aluminum thin films by electroless plating for applications in microbolometers

    Directory of Open Access Journals (Sweden)

    Do Ngoc Hieu

    2017-06-01

    Full Text Available An optimization process of electroless plating of nickel was carried out with NiCl2 as the nickel ion source, NaH2PO2 as the reduction agent, CH3COONa and Na3C6H5O7 as complexing agents. Electroless plated nickel layers on sputtered aluminum corning glass substrates with a resistivity of about 75.9 μΩ cm and a nickel concentration higher than 93% were obtained. This optimum process was successfully applied in growing nickel micropillars at selected areas with a well-controlled height. The microstructure of the masking layers was fabricated by means of optical photolithography for subsequent growth of nickel micropillars on selected areas. Micropillars size was defined by the opening size and the height was controlled by adjusting the plating time at a growth rate of 0.41 μm/min. This result shows that electroless nickel plating could be a good candidate for growing micropillars for applications in microbolometers.

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

    DEFF Research Database (Denmark)

    Dasmeh, Pouria; Serohijos, Adrian W R; Kepp, Kasper P

    2013-01-01

    Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10-20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the O2-binding affinity...... between Mb folding stability and protein abundance, suggesting that a selection pressure for stability acts proportionally to higher expression. We also identify a major divergence event leading to the common ancestor of whales, during which major stabilization occurred. Most of the positively selected...

  14. Enhanced binding by dextran-grafting to Protein A affinity chromatographic media.

    Science.gov (United States)

    Zhao, Lan; Zhu, Kai; Huang, Yongdong; Li, Qiang; Li, Xiunan; Zhang, Rongyue; Su, Zhiguo; Wang, Qibao; Ma, Guanghui

    2017-04-01

    Dextran-grafted Protein A affinity chromatographic medium was prepared by grafting dextran to agarose-based matrix, followed by epoxy-activation and Protein A coupling site-directed to sulfhydryl groups of cysteine molecules. An enhancement of both the binding performance and the stability was achieved for this dextran-grafted Protein A chromatographic medium. Its dynamic binding capacity was 61 mg immunoglobulin G/mL suction-dried gel, increased by 24% compared with that of the non-grafted medium. The binding capacity of dextran-grafted medium decreased about 7% after 40 cleaning-in-place cycles, much lower than that of the non-grafted medium as decreased about 15%. Confocal laser scanning microscopy results showed that immunoglobulin G was bound to both the outside and the inside of dextran-grafted medium faster than that of non-grafted one. Atomic force microscopy showed that this dextran-grafted Protein A medium had much rougher surface with a vertical coordinate range of ±80 nm, while that of non-grafted one was ±10 nm. Grafted dextran provided a more stereo surface morphology and immunoglobulin G molecules were more easily to be bound. This high-performance dextran-grafted Protein A affinity chromatographic medium has promising applications in large-scale antibody purification. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Use of Pd-Pt loaded graphene aerogel on nickel foam in direct ethanol fuel cell

    Science.gov (United States)

    Tsang, Chi Him A.; Leung, D. Y. C.

    2018-01-01

    A size customized binder-free bimetallic Pd-Pt loaded graphene aerogel deposited on nickel foam plate (Pd-Pt/GA/NFP) was prepared and used as an electrode for an alkaline direct ethanol fuel cell (DEFC) under room temperature. The effect of fuel concentration and metal composition on the output power density of the DEFC was systematically investigated. Under the optimum fuel concentration, the cell could achieve a value of 3.6 mW cm-2 at room temperature for the graphene electrode with Pd/Pt ratio approaching 1:1. Such results demonstrated the possibility of producing a size customized metal loaded GA/NFP electrode for fuel cell with high performance.

  16. Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

    Directory of Open Access Journals (Sweden)

    Sujatha P Koduvayur

    Full Text Available The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

  17. Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

    Science.gov (United States)

    Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

    2017-06-20

    Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

  18. Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

    Science.gov (United States)

    Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

    2015-02-01

    We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    International Nuclear Information System (INIS)

    Moaddel, Ruin; Wainer, Irving W.

    2006-01-01

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K d values) and non-linear chromatography can be used to assess the association (k on ) and dissociation (k off ) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein

  20. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    Energy Technology Data Exchange (ETDEWEB)

    Moaddel, Ruin [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States); Wainer, Irving W. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States)]. E-mail: Wainerir@grc.nia.nih.gov

    2006-03-30

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K {sub d} values) and non-linear chromatography can be used to assess the association (k {sub on}) and dissociation (k {sub off}) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein.

  1. Phase transformation in nickel during tribotesting

    Energy Technology Data Exchange (ETDEWEB)

    Hershberger, J. [Energy Technology Division, Argonne National Laboratory, Building 212 Room D204, 9700 S Cass Avenue, Argonne, IL 60439 (United States)]. E-mail: jhersh@anl.gov; Ajayi, O.O. [Energy Technology Division, Argonne National Laboratory, Building 212 Room D204, 9700 S Cass Avenue, Argonne, IL 60439 (United States); Fenske, G.R. [Energy Technology Division, Argonne National Laboratory, Building 212 Room D204, 9700 S Cass Avenue, Argonne, IL 60439 (United States)

    2005-12-15

    Commercially pure nickel was subjected to a polyalphaolefin-lubricated reciprocating tribotest with increasing load. A friction transition was observed and X-ray diffraction was performed on low-friction and high-friction areas. Hexagonal nickel or nickel carbide was formed at high friction. Broadening of the face-centered cubic peaks did not show dislocation structures characteristic of scuffing.

  2. Phase transformation in nickel during tribotesting

    International Nuclear Information System (INIS)

    Hershberger, J.; Ajayi, O.O.; Fenske, G.R.

    2005-01-01

    Commercially pure nickel was subjected to a polyalphaolefin-lubricated reciprocating tribotest with increasing load. A friction transition was observed and X-ray diffraction was performed on low-friction and high-friction areas. Hexagonal nickel or nickel carbide was formed at high friction. Broadening of the face-centered cubic peaks did not show dislocation structures characteristic of scuffing

  3. Communication between the Zinc and Nickel Sites in Dimeric HypA: Metal Recognition and pH Sensing

    Energy Technology Data Exchange (ETDEWEB)

    Herbst, R.; Perovic, I; Martin-Diaconescu, V; O’Brien, K; Chivers, P; Sondej Pochapsky, S; Pochapsky, T; Maroney, M

    2010-01-01

    Helicobacter pylori, a pathogen that colonizes the human stomach, requires the nickel-containing metalloenzymes urease and NiFe-hydrogenase to survive this low pH environment. The maturation of both enzymes depends on the metallochaperone, HypA. HypA contains two metal sites, an intrinsic zinc site and a low-affinity nickel binding site. X-ray absorption spectroscopy (XAS) shows that the structure of the intrinsic zinc site of HypA is dynamic and able to sense both nickel loading and pH changes. At pH 6.3, an internal pH that occurs during acid shock, the zinc site undergoes unprecedented ligand substitutions to convert from a Zn(Cys){sub 4} site to a Zn(His){sub 2}(Cys){sub 2} site. NMR spectroscopy shows that binding of Ni(II) to HypA results in paramagnetic broadening of resonances near the N-terminus. NOEs between the {beta}-CH{sub 2} protons of Zn cysteinyl ligands are consistent with a strand-swapped HypA dimer. Addition of nickel causes resonances from the zinc binding motif and other regions to double, indicating more than one conformation can exist in solution. Although the structure of the high-spin, 5-6 coordinate Ni(II) site is relatively unaffected by pH, the nickel binding stoichiometry is decreased from one per monomer to one per dimer at pH = 6.3. Mutation of any cysteine residue in the zinc binding motif results in a zinc site structure similar to that found for holo-WT-HypA at low pH and is unperturbed by the addition of nickel. Mutation of the histidines that flank the CXXC motifs results in a zinc site structure that is similar to holo-WT-HypA at neutral pH (Zn(Cys){sub 4}) and is no longer responsive to nickel binding or pH changes. Using an in vitro urease activity assay, it is shown that the recombinant protein is sufficient for recovery of urease activity in cell lysate from a HypA deletion mutant, and that mutations in the zinc-binding motif result in a decrease in recovered urease activity. The results are interpreted in terms of a model

  4. Communication between the Zinc and Nickel Sites in Dimeric HypA: Metal Recognition and pH Sensing

    International Nuclear Information System (INIS)

    Herbst, R.; Perovic, I.; Martin-Diaconescu, V.; O'Brien, K.; Chivers, P.; Sondej Pochapsky, S.; Pochapsky, T.; Maroney, M.

    2010-01-01

    Helicobacter pylori, a pathogen that colonizes the human stomach, requires the nickel-containing metalloenzymes urease and NiFe-hydrogenase to survive this low pH environment. The maturation of both enzymes depends on the metallochaperone, HypA. HypA contains two metal sites, an intrinsic zinc site and a low-affinity nickel binding site. X-ray absorption spectroscopy (XAS) shows that the structure of the intrinsic zinc site of HypA is dynamic and able to sense both nickel loading and pH changes. At pH 6.3, an internal pH that occurs during acid shock, the zinc site undergoes unprecedented ligand substitutions to convert from a Zn(Cys) 4 site to a Zn(His) 2 (Cys) 2 site. NMR spectroscopy shows that binding of Ni(II) to HypA results in paramagnetic broadening of resonances near the N-terminus. NOEs between the β-CH 2 protons of Zn cysteinyl ligands are consistent with a strand-swapped HypA dimer. Addition of nickel causes resonances from the zinc binding motif and other regions to double, indicating more than one conformation can exist in solution. Although the structure of the high-spin, 5-6 coordinate Ni(II) site is relatively unaffected by pH, the nickel binding stoichiometry is decreased from one per monomer to one per dimer at pH = 6.3. Mutation of any cysteine residue in the zinc binding motif results in a zinc site structure similar to that found for holo-WT-HypA at low pH and is unperturbed by the addition of nickel. Mutation of the histidines that flank the CXXC motifs results in a zinc site structure that is similar to holo-WT-HypA at neutral pH (Zn(Cys) 4 ) and is no longer responsive to nickel binding or pH changes. Using an in vitro urease activity assay, it is shown that the recombinant protein is sufficient for recovery of urease activity in cell lysate from a HypA deletion mutant, and that mutations in the zinc-binding motif result in a decrease in recovered urease activity. The results are interpreted in terms of a model wherein HypA controls the

  5. Peptides and Anti-peptide Antibodies for Small and Medium Scale Peptide and Anti-peptide Affinity Microarrays: Antigenic Peptide Selection, Immobilization, and Processing.

    Science.gov (United States)

    Zhang, Fan; Briones, Andrea; Soloviev, Mikhail

    2016-01-01

    This chapter describes the principles of selection of antigenic peptides for the development of anti-peptide antibodies for use in microarray-based multiplex affinity assays and also with mass-spectrometry detection. The methods described here are mostly applicable to small to medium scale arrays. Although the same principles of peptide selection would be suitable for larger scale arrays (with 100+ features) the actual informatics software and printing methods may well be different. Because of the sheer number of proteins/peptides to be processed and analyzed dedicated software capable of processing all the proteins and an enterprise level array robotics may be necessary for larger scale efforts. This report aims to provide practical advice to those who develop or use arrays with up to ~100 different peptide or protein features.

  6. Concurrent Working Memory Load Can Facilitate Selective Attention: Evidence for Specialized Load

    Science.gov (United States)

    Park, Soojin; Kim, Min-Shik; Chun, Marvin M.

    2007-01-01

    Load theory predicts that concurrent working memory load impairs selective attention and increases distractor interference (N. Lavie, A. Hirst, J. W. de Fockert, & E. Viding, see record 2004-17825-003). Here, the authors present new evidence that the type of concurrent working memory load determines whether load impairs selective attention or not.…

  7. Identification of Novel Surface-Exposed Proteins of Rickettsia rickettsii by Affinity Purification and Proteomics

    Science.gov (United States)

    Gong, Wenping; Xiong, Xiaolu; Qi, Yong; Jiao, Jun; Duan, Changsong; Wen, Bohai

    2014-01-01

    Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs) of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein) of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens. PMID:24950252

  8. Identification of novel surface-exposed proteins of Rickettsia rickettsii by affinity purification and proteomics.

    Directory of Open Access Journals (Sweden)

    Wenping Gong

    Full Text Available Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs, which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

  9. Affinity partitioning of human antibodies in aqueous two-phase systems.

    Science.gov (United States)

    Rosa, P A J; Azevedo, A M; Ferreira, I F; de Vries, J; Korporaal, R; Verhoef, H J; Visser, T J; Aires-Barros, M R

    2007-08-24

    The partitioning of human immunoglobulin (IgG) in a polymer-polymer and polymer-salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG-COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150-COOH and 40% (w/w) PEG 3350-COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350-COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150-COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150-COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.

  10. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  11. Cyclic fatigue resistance tests of Nickel-Titanium rotary files using simulated canal and weight loading conditions

    Directory of Open Access Journals (Sweden)

    Ok-In Cho

    2013-02-01

    Full Text Available Objectives This study compared the cyclic fatigue resistance of nickel-titanium (NiTi files obtained in a conventional test using a simulated canal with a newly developed method that allows the application of constant fatigue load conditions. Materials and Methods ProFile and K3 files of #25/.06, #30/.06, and #40/.04 were selected. Two types of testing devices were built to test their fatigue performance. The first (conventional device prescribed curvature inside a simulated canal (C-test, the second new device exerted a constant load (L-test whilst allowing any resulting curvature. Ten new instruments of each size and brand were tested with each device. The files were rotated until fracture and the number of cycles to failure (NCF was determined. The NCF were subjected to one-way ANOVA and Duncan's post-hoc test for each method. Spearman's rank correlation coefficient was computed to examine any association between methods. Results Spearman's rank correlation coefficient (ρ = -0.905 showed a significant negative correlation between methods. Groups with significant difference after the L-test divided into 4 clusters, whilst the C-test gave just 2 clusters. From the L-test, considering the negative correlation of NCF, K3 gave a significantly lower fatigue resistance than ProFile as in the C-test. K3 #30/.06 showed a lower fatigue resistance than K3 #25/.06, which was not found by the C-test. Variation in fatigue test methodology resulted in different cyclic fatigue resistance rankings for various NiTi files. Conclusions The new methodology standardized the load during fatigue testing, allowing determination fatigue behavior under constant load conditions.

  12. Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein.

    Science.gov (United States)

    Anjos, Liliana; Gomes, Ana S; Melo, Eduardo P; Canário, Adelino V; Power, Deborah M

    2013-03-01

    Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Residual Strain Characteristics of Nickel-coated FBG Sensors

    International Nuclear Information System (INIS)

    Cho, Won-Jae; Hwang, A-Reum; Kim, Sang-Woo

    2017-01-01

    A metal-coated FBG (fiber Bragg grating) sensor has a memory effect, which can recall the maximum strains experienced by the structure. In this study, a nickel-coated FBG sensor was fabricated through electroless (i.e., chemical plating) and electroplating. A thickness of approximately 43 μm of a nickel layer was achieved. Then, we conducted cyclic loading tests for the fabricated nickel-coated FBG sensors to verify their capability to produce residual strains. The results revealed that the residual strain induced by the nickel coating linearly increased with an increase in the maximum strain experienced by the sensor. Therefore, we verified that a nickel-coated FBG sensor has a memory effect. The fabrication methods and the results of the cycle loading test will provide basic information and guidelines in the design of a nickel-coated FBG sensor when it is applied in the development of structural health monitoring techniques.

  14. Residual Strain Characteristics of Nickel-coated FBG Sensors

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Won-Jae; Hwang, A-Reum; Kim, Sang-Woo [Hankyong National Univ., Ansung (Korea, Republic of)

    2017-07-15

    A metal-coated FBG (fiber Bragg grating) sensor has a memory effect, which can recall the maximum strains experienced by the structure. In this study, a nickel-coated FBG sensor was fabricated through electroless (i.e., chemical plating) and electroplating. A thickness of approximately 43 μm of a nickel layer was achieved. Then, we conducted cyclic loading tests for the fabricated nickel-coated FBG sensors to verify their capability to produce residual strains. The results revealed that the residual strain induced by the nickel coating linearly increased with an increase in the maximum strain experienced by the sensor. Therefore, we verified that a nickel-coated FBG sensor has a memory effect. The fabrication methods and the results of the cycle loading test will provide basic information and guidelines in the design of a nickel-coated FBG sensor when it is applied in the development of structural health monitoring techniques.

  15. Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein

    International Nuclear Information System (INIS)

    Rodriguez, K.; Talamantez, J.; Huang, W.; Reed, S.H.; Wang, Z.; Chen, L.; Feaver, W.J.; Friedberg, E.C.; Tomkinson, A.E.

    1998-01-01

    The nucleotide excision repair (NER) pathway of eukaryotes involves approximately 30 polypeptides. Reconstitution of this pathway with purified components is consistent with the sequential assembly of NER proteins at the DNA lesion. However, recent studies have suggested that NER proteins may be pre-assembled in a high molecular weight complex in the absence of DNA damage. To examine this model further, we have constructed a histidine-tagged version of the yeast DNA damage recognition protein Rad14. Affinity purification of this protein from yeast nuclear extracts resulted in the co-purification of Rad1, Rad7, Rad10, Rad16, Rad23, RPA, RPB1, and TFIIH proteins, whereas none of these proteins bound to the affinity resin in the absence of recombinant Rad14. Furthermore, many of the co-purifying proteins were present in approximately equimolar amounts. Co-elution of these proteins was also observed when the nuclear extract was fractionated by gel filtration, indicating that the NER proteins were associated in a complex with a molecular mass of >1000 kDa prior to affinity chromatography. The affinity purified NER complex catalyzed the incision of UV-irradiated DNA in an ATP-dependent reaction. We conclude that active high molecular weight complexes of NER proteins exist in undamaged yeast cells

  16. Displacement affinity chromatography of protein phosphatase one (PP1 complexes

    Directory of Open Access Journals (Sweden)

    Gourlay Robert

    2008-11-01

    Full Text Available Abstract Background Protein phosphatase one (PP1 is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.

  17. PLASS: Protein-ligand affinity statistical score a knowledge-based force-field model of interaction derived from the PDB

    Science.gov (United States)

    Ozrin, V. D.; Subbotin, M. V.; Nikitin, S. M.

    2004-04-01

    We have developed PLASS (Protein-Ligand Affinity Statistical Score), a pair-wise potential of mean-force for rapid estimation of the binding affinity of a ligand molecule to a protein active site. This scoring function is derived from the frequency of occurrence of atom-type pairs in crystallographic complexes taken from the Protein Data Bank (PDB). Statistical distributions are converted into distance-dependent contributions to the Gibbs free interaction energy for 10 atomic types using the Boltzmann hypothesis, with only one adjustable parameter. For a representative set of 72 protein-ligand structures, PLASS scores correlate well with the experimentally measured dissociation constants: a correlation coefficient R of 0.82 and RMS error of 2.0 kcal/mol. Such high accuracy results from our novel treatment of the volume correction term, which takes into account the inhomogeneous properties of the protein-ligand complexes. PLASS is able to rank reliably the affinity of complexes which have as much diversity as in the PDB.

  18. Effects of Gold Substrates on the Intrinsic and Extrinsic Activity of High-Loading Nickel-Based Oxyhydroxide Oxygen Evolution Catalysts

    DEFF Research Database (Denmark)

    Chakthranont, Pongkarn; Kibsgaard, Jakob; Gallo, Alessandro

    2017-01-01

    We systematically investigate the effects of Au substrates on the oxygen evolution activities of cathodically electrodeposited nickel oxyhydroxide (NiOOH), nickel–iron oxyhydroxide (NiFeOOH), and nickel–cerium oxyhydroxide (NiCeOOH) at varying loadings from 0 to 2000 nmol of metal/cm2. We determi...... high geometric current densities on flat substrates. By investigating the mass and site specific activities as a function of loading, we bridge the practical geometric activity to the fundamental intrinsic activity....

  19. Effect of two heavy metals, cadmium and nickel, on the organic load removal efficiency in a laboratory UASB reactor

    International Nuclear Information System (INIS)

    Forero, Luis Eduardo; Sierra, Jorge Humberto

    2004-01-01

    Experiments were carried out in three up flow anaerobic sludge blanket, UASB, reactors each with 3 L capacity, four hours of hydraulic retention time, (HRT) and volumetric organic load of 4,8 g/L/d. After the initial start phase, which was of 4.000 hours for the three reactors, they were affected in the following way: the first reactor was continuously feed with 5 mg/L of cadmium chloride, the second one was continuously feed with 10 mg/L of nickel chloride and the last one was not affected and served as reference. Efficiency in organic load removal was measured as oxygen chemical demand (OCD), the first reactor changed from 60% in the start phase (phase one) to 18% in the cadmium-affected phase (phase two), efficiency in removal (OCI) in reactor two varied from 60 to 24% and the last one did not change in a noticeable manner. Reactor one accumulated cadmium in the mud, whereas reactor two did not do that with nickel

  20. Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

    Science.gov (United States)

    Schnettler, Esther; Hemmes, Hans; Huismann, Rik; Goldbach, Rob; Prins, Marcel; Kormelink, Richard

    2010-11-01

    The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

  1. Peptide and protein loading into porous silicon wafers

    Energy Technology Data Exchange (ETDEWEB)

    Prestidge, C.A.; Barnes, T.J.; Mierczynska-Vasilev, A.; Kempson, I.; Peddie, F. [Ian Wark Research Institute, University of South Australia, Mawson Lakes (Australia); Barnett, C. [Medica Ltd, Malvern, Worcestershire, UK WR14 3SZ (United Kingdom)

    2008-02-15

    The influence of peptide/protein size and hydrophobicity on the physical and chemical aspects of loading within porous silicon (pSi) wafer samples has been determined using Atomic Force Microscopy (AFM) and Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS). Both Gramicidin A (a small hydrophobic peptide) and Papain (a larger hydrophilic protein) were observed (ToF-SIMS) to penetrate across the entire pSi layer, even at low loading levels. AFM surface imaging of pSi wafers during peptide/protein loading showed that surface roughness increased with Papain loading, but decreased with Gramicidin A loading. For Papain, the loading methodology was also found to influence loading efficiency. These differences indicate more pronounced surface adsorption of Papain. (copyright 2008 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  2. Preparation and characterization of fluorophenylboronic acid-functionalized affinity monolithic columns for the selective enrichment of cis-diol-containing biomolecules.

    Science.gov (United States)

    Li, Qianjin; Liu, Zhen

    2015-01-01

    Boronate affinity monolithic columns have been developed into an important means for the selective recognition and capture of cis-diol-containing biomolecules, such as glycoproteins, nucleosides and saccharides. The ligands of boronic acids are playing an important role in boronate affinity monolithic columns. Although several boronate affinity monoliths with high affinity toward cis-diol-containing biomolecules have been reported, only few publications are focused on their detailed procedures for preparation and characterization. This chapter describes in detail the preparation and characterization of a boronate affinity monolithic column applying 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA) as a ligand. The DFFPBA-functionalized monolithic column not only exhibited an ultrahigh boronate affinity toward cis-diol-containing biomolecules, but also showed great potential for the selective enrichment of cis-diol-containing biomolecules in real samples.

  3. Phosphatidylserine and Phosphatidylethanolamine Bind to Protein Z Cooperatively and with Equal Affinity.

    Directory of Open Access Journals (Sweden)

    Tanusree Sengupta

    Full Text Available Protein Z (PZ is an anticoagulant that binds with high affinity to Protein Z-dependent protease inhibitor (ZPI and accelerates the rate of ZPI-mediated inhibition of factor Xa (fXa by more than 1000-fold in the presence of Ca2+ and phospholipids. PZ promotion of the ZPI-fXa interaction results from the anchoring of the Gla domain of PZ onto phospholipid surfaces and positioning the bound ZPI in close proximity to the Gla-anchored fXa, forming a ternary complex of PZ/ZPI/fXa. Although interaction of PZ with phospholipid membrane appears to be absolutely crucial for its cofactor activity, little is known about the binding of different phospholipids to PZ. The present study was conceived to understand the interaction of different phospholipids with PZ. Experiments with both soluble lipids and model membranes revealed that PZ binds to phosphatidylserine (PS and phosphatidylethanolamine (PE with equal affinity (Kd~48 μM; further, PS and PE bound to PZ synergistically. Equilibrium dialysis experiments revealed two lipid-binding sites for both PS and PE. PZ binds with weaker affinity to other phospholipids, e.g., phosphatidic acid, phosphatidylglycerol, phosphatidylcholine and binding of these lipids is not synergistic with respect to PS. Both PS and PE -containing membranes supported the formation of a fXa-PZ complex. PZ protection of fXa from antithrombin inhibition were also shown to be comparable in presence of both PS: PC and PE: PC membranes. These findings are particularly important and intriguing since they suggest a special affinity of PZ, in vivo, towards activated platelets, the primary membrane involved in blood coagulation process.

  4. Scale factor management in the studies of affine models of shockproof garment elements

    Directory of Open Access Journals (Sweden)

    Denisov Oleg

    2018-01-01

    Full Text Available New samples of protective garment for performing construction work at height require numerous tests in conditions close to real conditions of extreme vital activity. The article presents some results of shockproof garment element studies and a description of a patented prototype. The tests were carried out on a model which geometric dimensions were convenient for manufacturing it in a limited batch. In addition, the used laboratory equipment (for example, a unique power pendulum, blanks made of a titanium-nickel alloy with a shape memory effect also imposed their limitations. The problem of the adequacy of the obtained experimental results transfer to mass-produced products was solved using tools of the classical similarity theory. Scale factor management influence in the affine modeling of the shockproof element, studied on the basis of the equiatomic titanium-nickel alloy with the shape memory effect, allowed us to assume, with a sufficient degree of reliability, the technical possibility of extrapolating the results of experimental studies to full-scale objects for the formation of the initial data of the mathematical model of shockproof garment dynamics elastoplastic deformation (while observing the similarity of the features of external loading.

  5. Scale factor management in the studies of affine models of shockproof garment elements

    Science.gov (United States)

    Denisov, Oleg; Pleshko, Mikhail; Ponomareva, Irina; Merenyashev, Vitaliy

    2018-03-01

    New samples of protective garment for performing construction work at height require numerous tests in conditions close to real conditions of extreme vital activity. The article presents some results of shockproof garment element studies and a description of a patented prototype. The tests were carried out on a model which geometric dimensions were convenient for manufacturing it in a limited batch. In addition, the used laboratory equipment (for example, a unique power pendulum), blanks made of a titanium-nickel alloy with a shape memory effect also imposed their limitations. The problem of the adequacy of the obtained experimental results transfer to mass-produced products was solved using tools of the classical similarity theory. Scale factor management influence in the affine modeling of the shockproof element, studied on the basis of the equiatomic titanium-nickel alloy with the shape memory effect, allowed us to assume, with a sufficient degree of reliability, the technical possibility of extrapolating the results of experimental studies to full-scale objects for the formation of the initial data of the mathematical model of shockproof garment dynamics elastoplastic deformation (while observing the similarity of the features of external loading).

  6. Plasma-polymerized films providing selective affinity to the polarity of vaporized organic solvents

    International Nuclear Information System (INIS)

    Akimoto, Takuo; Ikeshita, Yusuke; Terashima, Ryo; Karube, Isao

    2009-01-01

    Plasma-polymerized films (PPFs) were fabricated as recognition membranes for a vapor-sensing device, and their affinity to vaporized organic solvents was evaluated with surface plasmon resonance. The affinity we intended to create is the selective sorption of the vaporized organic solvents depending on their polarity. For this purpose, acetonitrile, ethylenediamine (EDA), styrene, hexamethyldisiloxane (HMDSO), and hexamethyldisilazane were used to fabricate PPFs. Vaporized methanol, ethanol, and 1-propanol were used as high-polar solvents to be analyzed. Hexane, toluene, and p-xylene were used as low-polar solvents. As a result, the HMDSO-PPF with 97.3 o of contact angle was found to provide affinity to the low-polar solvents. In contrast, the EDA-PPF with 7.1 o of contact angle provided affinity to the high-polar solvents. Observations of the surface morphology of the HMDSO- and EDA-PPFs with a scanning electron microscope revealed that they are composed of nano-scale islands.

  7. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.

    Science.gov (United States)

    Imaduwage, Kasun P; Go, Eden P; Zhu, Zhikai; Desaire, Heather

    2016-11-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have K i values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods. Graphical Abstract ᅟ.

  8. Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors.

    Science.gov (United States)

    Kamakoti, Vikramshankar; Panneer Selvam, Anjan; Radha Shanmugam, Nandhinee; Muthukumar, Sriram; Prasad, Shalini

    2016-07-18

    Molybdenum electrode based flexible biosensor on porous polyamide substrates has been fabricated and tested for its functionality as a protein affinity based biosensor. The biosensor performance was evaluated using a key cardiac biomarker; cardiac Troponin-I (cTnI). Molybdenum is a transition metal and demonstrates electrochemical behavior upon interaction with an electrolyte. We have leveraged this property of molybdenum for designing an affinity based biosensor using electrochemical impedance spectroscopy. We have evaluated the feasibility of detection of cTnI in phosphate-buffered saline (PBS) and human serum (HS) by measuring impedance changes over a frequency window from 100 mHz to 1 MHz. Increasing changes to the measured impedance was correlated to the increased dose of cTnI molecules binding to the cTnI antibody functionalized molybdenum surface. We achieved cTnI detection limit of 10 pg/mL in PBS and 1 ng/mL in HS medium. The use of flexible substrates for designing the biosensor demonstrates promise for integration with a large-scale batch manufacturing process.

  9. Single-step affinity purification for fungal proteomics.

    Science.gov (United States)

    Liu, Hui-Lin; Osmani, Aysha H; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B; De Souza, Colin P; Osmani, Stephen A

    2010-05-01

    A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

  10. Nickel and vanadium extraction from the Syrian petroleum coke

    International Nuclear Information System (INIS)

    Shlewit, H.; Alibrahim, M.

    2007-01-01

    Syrian petroleum coke samples were characterized and submitted for salt-roasting treatment in electric furnace to evaluate the convenience of this procedure for the extraction of the vanadium, nickel and sulfur from coke. Both solution and solid residue remaining after salt roasting were separated by filtration and were analyzed for vanadium, nickel and sulfur. The solution was analyzed by UV-Visible spectroscopy for vanadium and nickel and gravimetrically for sulfur. The solid residue and the untreated samples of petroleum coke were analyzed by XRF spectrometry. Results showed that more than 90% of sulfur and 60% of vanadium could be extracted by salt roasting treatment. An alternative procedure has been suggested, in which, more than 80% of sulfur and small percentage of vanadium can be leached by 0.75 M of Na 2 CO 3 solution at 70-80 Co. Vanadium was selectively extracted by DEHPA/TBP from the loaded leached solution. The extraction procedure flowsheet was also suggested. (authors)

  11. Analytical approaches for the characterization of nickel proteome.

    Science.gov (United States)

    Jiménez-Lamana, Javier; Szpunar, Joanna

    2017-08-16

    The use of nickel in modern industry and in consumer products implies some health problems for the human being. Nickel allergy and nickel carcinogenicity are well-known health effects related to human exposure to nickel, either during production of nickel-containing products or by direct contact with the final item. In this context, the study of nickel toxicity and nickel carcinogenicity involves the understanding of their molecular mechanisms and hence the characterization of the nickel-binding proteins in different biological samples. During the last 50 years, a broad range of analytical techniques, covering from the first chromatographic columns to the last generation mass spectrometers, have been used in order to fully characterize the nickel proteome. The aim of this review is to present a critical view of the different analytical approaches that have been applied for the purification, isolation, detection and identification of nickel-binding proteins. The different analytical techniques used are discussed from a critical point of view, highlighting advantages and limitations.

  12. The effect of ligation on the load deflection characteristics of nickel titanium orthodontic wire.

    Science.gov (United States)

    Kasuya, Shugo; Nagasaka, Satoshi; Hanyuda, Ai; Ishimura, Sadao; Hirashita, Ayao

    2007-12-01

    This study examined the effect of ligation on the load-deflection characteristics of nickel-titanium (NiTi) orthodontic wire. A modified three-point bending system was used for bending the NiTi round wire, which was inserted and ligated in the slots of three brackets, one of which was bonded to each of the three bender rods. Three different ligation methods, stainless steel ligature (SSL), slot lid (SL), and elastomeric ligature (EL), were employed, as well as a control with neither bracket nor ligation (NBL). The tests were repeated five times under each condition. Comparisons were made of load-deflection curve, load at maximum deflection of 2,000 microm, and load at a deflection of 1,500 microm during unloading. Analysis of Variance (ANOVA) and Dunnett's test were conducted to determine method difference (alpha = 0.05). The interaction between deflection and ligation was tested, using repeated-measures ANOVA (alpha = 0.05). The load values of the ligation groups were two to three times greater than the NBL group at a deflection of 1,500 microm during unloading: 4.37 N for EL, 3.90 N for SSL, 3.02 N for SL, and 1.49 N for NBL (P wire may make NiTi wire exhibit a significantly heavier load than that traditionally expected. NiTi wire exhibited the majority of its true superelasticity with SL, whereas EL may act as a restraint on its superelasticity.

  13. Catalytic CO Oxidation over Au Nanoparticles Loaded Nanoporous Nickel Phosphate Composite

    Directory of Open Access Journals (Sweden)

    Xiaonan Leng

    2015-01-01

    Full Text Available Au/nickel phosphate-5 (Au/VSB-5 composite with the noble metal loading amount of 1.43 wt.% is prepared by using microporous VSB-5 nanocrystals as the support. Carbon monoxide (CO oxidation reaction is carried out over the sample with several catalytic cycles. Complete conversion of CO is achieved at 238°C over the catalyst at the first catalytic cycle. The catalytic activity improved greatly at the second cycle with the complete conversion fulfilled at 198°C and preserved for the other cycles. A series of experiments such as X-ray diffraction (XRD, high resolution transmission electron microscopy (HRTEM, ultraviolet-visible (UV-vis spectroscopy, and X-ray photoelectron spectroscopy (XPS are carried out to characterize the catalysts before and after the reaction to study the factors influencing this promotion at the second cycle.

  14. The biocompatibility of dense and porous Nickel-Titanium produced by selective laser melting

    Energy Technology Data Exchange (ETDEWEB)

    Habijan, T., E-mail: Tim.Habijan@rub.de [Surgical Research, Department of Surgery, BG Kliniken Bergmannsheil, Ruhr-University Bochum, Buerkle-de-la-Camp-Platz 1, 44789 Bochum (Germany); Haberland, C.; Meier, H. [Institute Product and Service Engineering, Ruhr-University Bochum (Germany); Frenzel, J. [Institute for Materials, Ruhr-University Bochum (Germany); Wittsiepe, J. [Department of Hygiene, Social and Environmental Medicine, Ruhr-University Bochum (Germany); Wuwer, C.; Greulich, C.; Schildhauer, T.A.; Koeller, M. [Surgical Research, Department of Surgery, BG Kliniken Bergmannsheil, Ruhr-University Bochum, Buerkle-de-la-Camp-Platz 1, 44789 Bochum (Germany)

    2013-01-01

    Nickel-Titanium shape memory alloys (NiTi-SMA) are of biomedical interest due to their unusual range of pure elastic deformability and their elastic modulus, which is closer to that of bone than any other metallic or ceramic material. Newly developed porous NiTi, produced by Selective Laser Melting (SLM), is currently under investigation as a potential carrier material for human mesenchymal stem cells (hMSC). SLM enables the production of highly complex and tailor-made implants for patients on the basis of CT data. Such implants could be used for the reconstruction of the skull, face, or pelvis. hMSC are a promising cell type for regenerative medicine and tissue engineering due to their ability to support the regeneration of critical size bone defects. Loading porous SLM-NiTi implants with autologous hMSC may enhance bone growth and healing for critical bone defects. The purpose of this study was to assess whether porous SLM-NiTi is a suitable carrier for hMSC. Specimens of varying porosity and surface structure were fabricated via SLM. hMSC were cultured for 8 days on NiTi specimens, and cell viability was analyzed using two-color fluorescence staining. Viable cells were detected on all specimens after 8 days of cell culture. Cell morphology and surface topography were analyzed by scanning electron microscopy (SEM). Cell morphology and surface topology were dependent on the orientation of the specimens during SLM production. The Nickel ion release can be reduced significantly by aligned laser processing conditions. The presented results clearly attest that both dense SLM-NiTi and porous SLM-NiTi are suitable carriers for hMSC. Nevertheless, before carrying out in vivo studies, some work on optimization of the manufacturing process and post-processing is required. - Highlights: Black-Right-Pointing-Pointer Specimens, varying in porosity and surface structure were produced via SLM. Black-Right-Pointing-Pointer Biocompatibility of these specimens was analyzed. Black

  15. Synthetic Polymer Affinity Ligand for Bacillus thuringiensis ( Bt) Cry1Ab/Ac Protein: The Use of Biomimicry Based on the Bt Protein-Insect Receptor Binding Mechanism.

    Science.gov (United States)

    Liu, Mingming; Huang, Rong; Weisman, Adam; Yu, Xiaoyang; Lee, Shih-Hui; Chen, Yalu; Huang, Chao; Hu, Senhua; Chen, Xiuhua; Tan, Wenfeng; Liu, Fan; Chen, Hao; Shea, Kenneth J

    2018-05-24

    We report a novel strategy for creating abiotic Bacillus thuringiensis ( Bt) protein affinity ligands by biomimicry of the recognition process that takes place between Bt Cry1Ab/Ac proteins and insect receptor cadherin-like Bt-R 1 proteins. Guided by this strategy, a library of synthetic polymer nanoparticles (NPs) was prepared and screened for binding to three epitopes 280 FRGSAQGIEGS 290 , 368 RRPFNIGINNQQ 379 and 436 FRSGFSNSSVSIIR 449 located in loop α8, loop 2 and loop 3 of domain II of Bt Cry1Ab/Ac proteins. A negatively charged and hydrophilic nanoparticle (NP12) was found to have high affinity to one of the epitopes, 368 RRPFNIGINNQQ 379 . This same NP also had specific binding ability to both Bt Cry1Ab and Bt Cry1Ac, proteins that share the same epitope, but very low affinity to Bt Cry2A, Bt Cry1C and Bt Cry1F closely related proteins that lack epitope homology. To locate possible NP- Bt Cry1Ab/Ac interaction sites, NP12 was used as a competitive inhibitor to block the binding of 865 NITIHITDTNNK 876 , a specific recognition site in insect receptor Bt-R 1 , to 368 RRPFNIGINNQQ 379 . The inhibition by NP12 reached as high as 84%, indicating that NP12 binds to Bt Cry1Ab/Ac proteins mainly via 368 RRPFNIGINNQQ 379 . This epitope region was then utilized as a "target" or "bait" for the separation and concentration of Bt Cry1Ac protein from the extract of transgenic Bt cotton leaves by NP12. This strategy, based on the antigen-receptor recognition mechanism, can be extended to other biotoxins and pathogen proteins when designing biomimic alternatives to natural protein affinity ligands.

  16. A Novel Affinity Tag, ABTAG, and Its Application to the Affinity Screening of Single-Domain Antibodies Selected by Phage Display

    Directory of Open Access Journals (Sweden)

    Greg Hussack

    2017-10-01

    Full Text Available ABTAG is a camelid single-domain antibody (sdAb that binds to bovine serum albumin (BSA with low picomolar affinity. In surface plasmon resonance (SPR analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6 sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.

  17. Oxygen-induced intergranular fracture of the nickel-base alloy IN718 during mechanical loading at high temperatures

    Directory of Open Access Journals (Sweden)

    Krupp Ulrich

    2004-01-01

    Full Text Available There is a transition in the mechanical-failure behavior of nickel-base superalloys from ductile transgranular crack propagation to time-dependent intergranular fracture when the temperature exceeds about 600 °C. This transition is due to oxygen diffusion into the stress field ahead of the crack tip sufficient to cause brittle decohesion of the grain boundaries. Since very high cracking rates were observed during fixed-displacement loading of IN718, it is not very likely that grain boundary oxidation governs the grain-boundary-separation process, as has been proposed in several studies on the fatigue-damage behavior of the nickel-base superalloy IN718. Further studies on bicrystal and thermomechanically processed specimens of IN718 have shown that this kind of brittle fracture, which has been termed "dynamic embrittlement", depends strongly on the structure of the grain boundaries.

  18. Low-cost removal of organic pollutants with nickel nanoparticle loaded ordered macroporous hydrogel as high performance catalyst

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Mingyi, E-mail: tmyi@tjcu.edu.cn [Department of Applied Chemistry, School of Science, Tianjin University of Commerce, Tianjin 300134 (China); Huang, Guanbo, E-mail: gbhuang2007@hotmail.com [Department of Chemistry, School of Science, Tianjin University, Tianjin 300072 (China); Zhang, Sai [Department of Applied Chemistry, School of Science, Tianjin University of Commerce, Tianjin 300134 (China); Liu, Yue [Department of Chemistry, School of Science, Tianjin University, Tianjin 300072 (China); Li, Xianxian [Department of Applied Chemistry, School of Science, Tianjin University of Commerce, Tianjin 300134 (China); Wang, Xingrui [Department of Chemistry, School of Science, Tianjin University, Tianjin 300072 (China); Pang, Xiaobo [Department of Applied Chemistry, School of Science, Tianjin University of Commerce, Tianjin 300134 (China); Qiu, Haixia, E-mail: qhx@tju.edu.cn [Department of Chemistry, School of Science, Tianjin University, Tianjin 300072 (China)

    2014-06-01

    A facile route for the in situ preparation of catalytically active Ni nanoparticles (NPs) in ordered macroporous hydrogel (OMH) has been developed. The hydrogel was fabricated based on polystyrene colloid template. The electronegativity of amide and carboxyl groups on the poly(acrylamide-co-acryl acid) chains of the hydrogel caused strong binding of Ni{sup 2+} ions which made them distribute uniformly inside the hydrogel. When immersed in NaBH{sub 4} aqueous solution, the Ni{sup 2+} ions on the hydrogel were reduced to Ni NPs. The resultant Ni NPs loaded OMH showed good catalytic activity for the reduction of a common organic pollutant, 4-nitrophenol, with NaBH{sub 4}. A kinetic study of the catalytic reaction was carried out. The rate constant per unit weight could reach 0.53 s{sup −1} g{sup −1}, which is much better than many common hydrogel loaded nickel catalysts. Moreover, the current catalyst can be easily separated and recovered with stable catalytic activity. - Highlights: • A new poly(acrylamide-co-acryl acid) hydrogel with ordered macropores. • A simple in situ fabrication of nickel nanoparticles under mild conditions. • High-performance heterogeneous catalyst for removal of nitrophenol from water. • Good recyclability of catalyst without any complicated regeneration process.

  19. Low-cost removal of organic pollutants with nickel nanoparticle loaded ordered macroporous hydrogel as high performance catalyst

    International Nuclear Information System (INIS)

    Tang, Mingyi; Huang, Guanbo; Zhang, Sai; Liu, Yue; Li, Xianxian; Wang, Xingrui; Pang, Xiaobo; Qiu, Haixia

    2014-01-01

    A facile route for the in situ preparation of catalytically active Ni nanoparticles (NPs) in ordered macroporous hydrogel (OMH) has been developed. The hydrogel was fabricated based on polystyrene colloid template. The electronegativity of amide and carboxyl groups on the poly(acrylamide-co-acryl acid) chains of the hydrogel caused strong binding of Ni 2+ ions which made them distribute uniformly inside the hydrogel. When immersed in NaBH 4 aqueous solution, the Ni 2+ ions on the hydrogel were reduced to Ni NPs. The resultant Ni NPs loaded OMH showed good catalytic activity for the reduction of a common organic pollutant, 4-nitrophenol, with NaBH 4 . A kinetic study of the catalytic reaction was carried out. The rate constant per unit weight could reach 0.53 s −1  g −1 , which is much better than many common hydrogel loaded nickel catalysts. Moreover, the current catalyst can be easily separated and recovered with stable catalytic activity. - Highlights: • A new poly(acrylamide-co-acryl acid) hydrogel with ordered macropores. • A simple in situ fabrication of nickel nanoparticles under mild conditions. • High-performance heterogeneous catalyst for removal of nitrophenol from water. • Good recyclability of catalyst without any complicated regeneration process

  20. JAEA thermodynamic database for performance assessment of geological disposal of high-level and TRU wastes. Selection of thermodynamic data of cobalt and nickel

    International Nuclear Information System (INIS)

    Kitamura, Akira; Yui, Mikazu; Kirishima, Akira; Saito, Takumi; Shibutani, Sanae; Tochiyama, Osamu

    2009-11-01

    Within the scope of the JAEA thermodynamic database project for performance assessment of geological disposal of high-level and TRU wastes, the selection of the thermodynamic data on the inorganic compounds and complexes of cobalt and nickel have been carried out. For cobalt, extensive literature survey has been performed and all the obtained literatures have been carefully reviewed to select the thermodynamic data. Selection of thermodynamic data of nickel has been based on a thermodynamic database published by the Nuclear Energy Agency in the Organisation for Economic Co-operation and Development (OECD/NEA), which has been carefully reviewed by the authors, and then thermodynamic data have been selected after surveying latest literatures. Based on the similarity of chemical properties between cobalt and nickel, complementary thermodynamic data of nickel and cobalt species expected under the geological disposal condition have been selected to complete the thermodynamic data set for the performance assessment of geological disposal of radioactive wastes. (author)

  1. Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

    Science.gov (United States)

    Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

    2015-06-01

    Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

  2. Contributions of pocket depth and electrostatic interactions to affinity and selectivity of receptors for methylated lysine in water.

    Science.gov (United States)

    Beaver, Joshua E; Peacor, Brendan C; Bain, Julianne V; James, Lindsey I; Waters, Marcey L

    2015-03-21

    Dynamic combinatorial chemistry was used to generate a set of receptors for peptides containing methylated lysine (KMen, n = 0-3) and study the contribution of electrostatic effects and pocket depth to binding affinity and selectivity. We found that changing the location of a carboxylate resulted in an increase in preference for KMe2, presumably based on ability to form a salt bridge with KMe2. The number of charged groups on either the receptor or peptide guest systematically varied the binding affinities to all guests by approximately 1-1.5 kcal mol(-1), with little influence on selectivity. Lastly, formation of a deeper pocket led to both increased affinity and selectivity for KMe3 over the lower methylation states. From these studies, we identified that the tightest binder was a receptor with greater net charge, with a Kd of 0.2 μM, and the receptor with the highest selectivity was the one with the deepest pocket, providing 14-fold selectivity between KMe3 and KMe2 and a Kd for KMe3 of 0.3 μM. This work provides key insights into approaches to improve binding affinity and selectivity in water, while also demonstrating the versatility of dynamic combinatorial chemistry for rapidly exploring the impact of subtle changes in receptor functionality on molecular recognition in water.

  3. Affinity-Selected Filamentous Bacteriophage as a Probe for Acoustic Wave Biodetectors of Salmonella typhimurium

    National Research Council Canada - National Science Library

    Olsen, Eric V; Sorokulova, Iryna B; Petrenko, Valery A; Chen, I-Hsuan; Barbaree, James M; Vodyanoy, Vitaly J

    2005-01-01

    Proof-in-concept biosensors were prepared for the rapid detection of Salmonella typhimurium in solution, based on affinity-selected filamentous phage prepared as probes physically adsorbed to piezoelectric transducers...

  4. The role of nickel in urea assimilation by algae.

    Science.gov (United States)

    Rees, T A; Bekheet, I A

    1982-12-01

    Nickel is required for urease synthesis by Phaeodactylum tricornutum and Tetraselmis subcordiformis and for growth on urea by Phaeodactylum. There is no requirement for nickel for urea amidolyase synthesis by Chlorella fusca var. vacuolata. Neither copper nor palladium can substitute for nickel but cobalt partially restored urease activity in Phaeodactylum. The addition of nickel to nickel-deficient cultures of Phaeodactylum or Tetraselmis resulted in a rapid increase of urease activity to 7-30 times the normal level; this increase was not inhibited by cycloheximide. It is concluded that nickel-deficient cells over-produce a non-functional urease protein and that either nickel or the functional urease enzyme participates in the regulation of the production of urease protein.

  5. The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

    Science.gov (United States)

    Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

    2012-09-01

    Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses. © 2012 John Wiley & Sons A/S.

  6. Comparison of different transition metal ions for immobilized metal affinity chromatography of selenoprotein P from human plasma

    DEFF Research Database (Denmark)

    Sidenius, U; Farver, O; Jøns, O

    1999-01-01

    Cu2+, Ni2+, Zn2+, Co2+ and Cd2+ were evaluated in metal ion affinity chromatography for enrichment of selenoprotein P, and immobilized Co2+ affinity chromatography was found to be the most selective chromatographic method. The chromatography was performed by fast protein liquid chromatography...

  7. Is Hand Selection Modulated by Cognitive-perceptual Load?

    Science.gov (United States)

    Liang, Jiali; Wilkinson, Krista; Sainburg, Robert L

    2018-01-15

    Previous studies proposed that selecting which hand to use for a reaching task appears to be modulated by a factor described as "task difficulty". However, what features of a task might contribute to greater or lesser "difficulty" in the context of hand selection decisions has yet to be determined. There has been evidence that biomechanical and kinematic factors such as movement smoothness and work can predict patterns of selection across the workspace, suggesting a role of predictive cost analysis in hand-selection. We hypothesize that this type of prediction for hand-selection should recruit substantial cognitive resources and thus should be influenced by cognitive-perceptual loading. We test this hypothesis by assessing the role of cognitive-perceptual loading on hand selection decisions, using a visual search task that presents different levels of difficulty (cognitive-perceptual load), as established in previous studies on overall response time and efficiency of visual search. Although the data are necessarily preliminary due to small sample size, our data suggested an influence of cognitive-perceptual load on hand selection, such that the dominant hand was selected more frequently as cognitive load increased. Interestingly, cognitive-perceptual loading also increased cross-midline reaches with both hands. Because crossing midline is more costly in terms of kinematic and kinetic factors, our findings suggest that cognitive processes are normally engaged to avoid costly actions, and that the choice not-to-cross midline requires cognitive resources. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  8. The biocompatibility of dense and porous Nickel-Titanium produced by selective laser melting.

    Science.gov (United States)

    Habijan, T; Haberland, C; Meier, H; Frenzel, J; Wittsiepe, J; Wuwer, C; Greulich, C; Schildhauer, T A; Köller, M

    2013-01-01

    Nickel-Titanium shape memory alloys (NiTi-SMA) are of biomedical interest due to their unusual range of pure elastic deformability and their elastic modulus, which is closer to that of bone than any other metallic or ceramic material. Newly developed porous NiTi, produced by Selective Laser Melting (SLM), is currently under investigation as a potential carrier material for human mesenchymal stem cells (hMSC). SLM enables the production of highly complex and tailor-made implants for patients on the basis of CT data. Such implants could be used for the reconstruction of the skull, face, or pelvis. hMSC are a promising cell type for regenerative medicine and tissue engineering due to their ability to support the regeneration of critical size bone defects. Loading porous SLM-NiTi implants with autologous hMSC may enhance bone growth and healing for critical bone defects. The purpose of this study was to assess whether porous SLM-NiTi is a suitable carrier for hMSC. Specimens of varying porosity and surface structure were fabricated via SLM. hMSC were cultured for 8 days on NiTi specimens, and cell viability was analyzed using two-color fluorescence staining. Viable cells were detected on all specimens after 8 days of cell culture. Cell morphology and surface topography were analyzed by scanning electron microscopy (SEM). Cell morphology and surface topology were dependent on the orientation of the specimens during SLM production. The Nickel ion release can be reduced significantly by aligned laser processing conditions. The presented results clearly attest that both dense SLM-NiTi and porous SLM-NiTi are suitable carriers for hMSC. Nevertheless, before carrying out in vivo studies, some work on optimization of the manufacturing process and post-processing is required. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Protein Scaffolding for Small Molecule Catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Baker, David [Univ. of Washington, Seattle, WA (United States)

    2014-09-14

    We aim to design hybrid catalysts for energy production and storage that combine the high specificity, affinity, and tunability of proteins with the potent chemical reactivities of small organometallic molecules. The widely used Rosetta and RosettaDesign methodologies will be extended to model novel protein / small molecule catalysts in which one or many small molecule active centers are supported and coordinated by protein scaffolding. The promise of such hybrid molecular systems will be demonstrated with the nickel-phosphine hydrogenase of DuBois et. al.We will enhance the hydrogenase activity of the catalyst by designing protein scaffolds that incorporate proton relays and systematically modulate the local environment of the catalyticcenter. In collaboration with DuBois and Shaw, the designs will be experimentally synthesized and characterized.

  10. A novel strategy using MASCOT Distiller for analysis of cleavable isotope-coded affinity tag data to quantify protein changes in plasma.

    Science.gov (United States)

    Leung, Kit-Yi; Lescuyer, Pierre; Campbell, James; Byers, Helen L; Allard, Laure; Sanchez, Jean-Charles; Ward, Malcolm A

    2005-08-01

    A novel strategy consisting of cleavable Isotope-Coded Affinity Tag (cICAT) combined with MASCOT Distiller was evaluated as a tool for the quantification of proteins in "abnormal" patient plasma, prepared by pooling samples from patients with acute stroke. Quantification of all light and heavy cICAT-labelled peptide ion pairs was obtained using MASCOT Distiller combined with a proprietary software. Peptides displaying differences were selected for identification by MS. These preliminary results show the promise of our approach to identify potential biomarkers.

  11. Direct electrochemistry and electrocatalysis of heme proteins immobilised in carbon-coated nickel magnetic nanoparticle-chitosan-dimethylformamide composite films in room-temperature ionic liquids.

    Science.gov (United States)

    Wang, Ting; Wang, Lu; Tu, Jiaojiao; Xiong, Huayu; Wang, Shengfu

    2013-12-01

    The direct electrochemistry and electrocatalysis of heme proteins entrapped in carbon-coated nickel magnetic nanoparticle-chitosan-dimethylformamide (CNN-CS-DMF) composite films were investigated in the hydrophilic ionic liquid [bmim][BF4]. The surface morphologies of a representative set of films were characterised via scanning electron microscopy. The proteins immobilised in the composite films were shown to retain their native secondary structure using UV-vis spectroscopy. The electrochemical performance of the heme proteins-CNN-CS-DMF films was evaluated via cyclic voltammetry and chronoamperometry. A pair of stable and well-defined redox peaks was observed for the heme protein films at formal potentials of -0.151 V (HRP), -0.167 V (Hb), -0.155 V (Mb) and -0.193 V (Cyt c) in [bmim][BF4]. Moreover, several electrochemical parameters of the heme proteins were calculated by nonlinear regression analysis of the square-wave voltammetry. The addition of CNN significantly enhanced not only the electron transfer of the heme proteins but also their electrocatalytic activity toward the reduction of H2O2. Low apparent Michaelis-Menten constants were obtained for the heme protein-CNN-CS-DMF films, demonstrating that the biosensors have a high affinity for H2O2. In addition, the resulting electrodes displayed a low detection limit and improved sensitivity for detecting H2O2, which indicates that the biocomposite film can serve as a platform for constructing new non-aqueous biosensors for real detection. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Non-affine deformation in microstructure selection in solids: I. Molecular dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharya, Jayee; Paul, Arya; Sengupta, Surajit [S N Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Calcutta 700 098 (India); Rao, Madan [Raman Research Institute, C V Raman Avenue, Bangalore 560 080 (India)

    2008-09-10

    We study the nucleation dynamics and microstructure selection in a model two-dimensional solid undergoing a square to rhombic transformation, using coarse-grained molecular dynamics (MD) simulations. We find a range of microstructures depending on the depth of quench. The transformations are accompanied by the creation of transient and localized non-affine zones (NAZ), which evolve with the rapidly moving parent-product interface. These plastic regions are created beyond a threshold stress, at a rate proportional to the local stress. We show that the dynamics of NAZs determines the selection of microstructure, including the ferrite and martensite.

  13. Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

    Science.gov (United States)

    Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2017-04-01

    Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Recombinant spider silk genetically functionalized with affinity domains.

    Science.gov (United States)

    Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

    2014-05-12

    Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

  15. PANDA: Protein function prediction using domain architecture and affinity propagation.

    Science.gov (United States)

    Wang, Zheng; Zhao, Chenguang; Wang, Yiheng; Sun, Zheng; Wang, Nan

    2018-02-22

    We developed PANDA (Propagation of Affinity and Domain Architecture) to predict protein functions in the format of Gene Ontology (GO) terms. PANDA at first executes profile-profile alignment algorithm to search against PfamA, KOG, COG, and SwissProt databases, and then launches PSI-BLAST against UniProt for homologue search. PANDA integrates a domain architecture inference algorithm based on the Bayesian statistics that calculates the probability of having a GO term. All the candidate GO terms are pooled and filtered based on Z-score. After that, the remaining GO terms are clustered using an affinity propagation algorithm based on the GO directed acyclic graph, followed by a second round of filtering on the clusters of GO terms. We benchmarked the performance of all the baseline predictors PANDA integrates and also for every pooling and filtering step of PANDA. It can be found that PANDA achieves better performances in terms of area under the curve for precision and recall compared to the baseline predictors. PANDA can be accessed from http://dna.cs.miami.edu/PANDA/ .

  16. Excessive nickel release from mobile phones--a persistent cause of nickel allergy and dermatitis.

    Science.gov (United States)

    Jensen, Peter; Johansen, Jeanne D; Zachariae, Claus; Menné, Torkil; Thyssen, Jacob P

    2011-12-01

    Despite the political intention to limit nickel allergy and dermatitis in Europeans, nickel allergy remains frequent. There are several explanations for the persistence of nickel allergy and dermatitis, including the increasing use of mobile phones. Before regulation of nickel release from mobile phones, we showed that eight (19.5%) of 41 mobile phones marketed in Denmark between 2003 and 2007 released nickel in concentrations that may result in nickel allergy and dermatitis. In 2009, the EU Nickel Directive was revised to include nickel-releasing mobile phones. To investigate the proportion of mobile phones sold in Denmark that release nickel after regulation. Metallic parts from 50 randomly selected mobile phones currently for sale in Denmark were tested for nickel release by use of the dimethylglyoxime (DMG)-nickel spot test. Nine (18%) phones showed at least one positive DMG test reaction and two phones had more than one DMG test-positive spot. Apparently, the proportion of mobile phones with significant nickel release remains unchanged, despite the 2009 revision of the EU Nickel Directive. We encourage manufacturers to measure nickel release from metallic components used in the assembly of mobile phones to ensure safe products. © 2011 John Wiley & Sons A/S.

  17. A simple and selective method for the separation of Cu radioisotopes from nickel

    Energy Technology Data Exchange (ETDEWEB)

    Fan Xianfeng [School of Physics and Astronomy, University of Birmingham, B15 2TT Birmingham (United Kingdom)]. E-mail: x.fan@bham.ac.uk; Parker, David J. [School of Physics and Astronomy, University of Birmingham, B15 2TT Birmingham (United Kingdom); Smith, Mike D. [School of Physics and Astronomy, University of Birmingham, B15 2TT Birmingham (United Kingdom); Ingram, Andy [School of Physics and Astronomy, University of Birmingham, B15 2TT Birmingham (United Kingdom); Yang Zhufang [School of Physics and Astronomy, University of Birmingham, B15 2TT Birmingham (United Kingdom); Seville, Jonathan P.K. [School of Physics and Astronomy, University of Birmingham, B15 2TT Birmingham (United Kingdom)

    2006-10-15

    Separation of copper radioisotopes from a nickel target is normally performed using solvent extraction or anion exchange rather than using cationic exchange. A commonly held opinion is that cationic exchangers have very similar thermodynamic complexation constants for metallic ions with identical charges, therefore making the separation very difficult or impossible. The results presented in this article indicate that the selectivity of Chelex-100 (a cationic ion exchanger) for Cu radioisotope and Ni ions not only depends on the thermodynamic complexation constant in the resin but also markedly varies with the concentration of mobile H{sup +}. In our developed method, separation of copper radioisotopes from a nickel target was fulfilled in a column filled with Chelex-100 via controlling the HNO{sub 3} concentration of the eluent, and the separation is much more effective, simple and economical in comparison with the common method of anion exchange. For an irradiated nickel target with 650 mg Ni, after separation, the loss of Cu radioisotopes in the nickel portion was reduced from 30% to 0.33% of the total initial radioactivity and the nickel mixed into the radioactive products was reduced from 9.5 to 0.5 mg. This significant improvement will make subsequent labeling much easier and reduce consumption of chelating agents and other chemicals during labeling. If the labeled agent is used in human medical applications, the developed method will significantly decrease the uptake of Ni and chelating agents by patients, therefore reducing both the stress on human body associated with clearing the chemicals from blood and tissue and the risk of various types of acute and chronic disorder due to exposure to Ni.

  18. The cost of nickel allergy

    DEFF Research Database (Denmark)

    Hamann, Carsten R; Hamann, Dathan; Hamann, Curtis

    2013-01-01

    %), followed by aluminium-bronze (62, 17%). In total, 239 denominations released nickel (28%). Coins from Bolivia, Brazil and Costa Rica did not release nickel. Fewer than one-third of the denominations or issues from China, India, the euro area and Indonesia released nickel. In the United States, the Russian...... Federation, Japan, and Mexico, one-third or more of the denominations released nickel. Conclusions. This worldwide selection of circulating coins covered countries with 75% of the world population, and shows that the majority of the world population lives in countries where coins release nickel. Pertinently...

  19. Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

    Science.gov (United States)

    Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

    2016-04-05

    Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods.

  20. Microscopic Features of Fractured Fragment of Nickel-Titanium Endodontic Instruments by Two Different Modes of Torsional Loading

    Directory of Open Access Journals (Sweden)

    Ibrahim H. Abu-Tahun

    2018-01-01

    Full Text Available This study compared the microscopic features of the fractured endodontic nickel-titanium (NiTi rotary instruments by two different torsional loadings: repetitive torsional loading (RTL and single torsional loading (STL based on the International Organization for Standardization (ISO. ProTaper Next, HyFlex EDM, and V-Taper 2 were compared in this study. In the STL method, the torsional load was applied after fixing the 3 mm tip of the file, by continuous clockwise rotation (2 rpm until fracture. In the RTL method, a preset rotational loading (0.5 N·cm was applied and the clockwise loading to the preset torque and counterclockwise unloading to original position were repeated at 50 rpm until the file fractured. Fractured fragments by two methods were compared under a scanning electron microscope (SEM to examine the topographic features of the fractured surfaces and longitudinal aspects. SEM examinations showed significantly different features according to the loading methods. Specimens from the RTL method showed ruptured aspects on cross sections, with multiple areas of initiated cracks while the STL method showed the typical features of torsional failure, such as circular abrasion marks and fatigue dimples. This study suggested a new repetitive torsional loading method which is much more clinically relevant and may result in a different fracture feature from STL method.

  1. hPEPT1 Affinity and Translocation of Selected Gln-Sar and Glu-Sar Dipeptide Derivatives

    DEFF Research Database (Denmark)

    Eriksson, A. H.; Elm, Peter L.; Begtrup, Mikael

    2005-01-01

    using 14C-labeled Gly-Sar. Translocation was measured as fluorescence ratios induced by the substrates using the fluorescent probe BCECF and an epifluorescence microscope setup. All compounds showed high affinity to hPEPT1, but only the amides l-Gln(N,N-dimethyl)-Sar and l-Gln(N-piperidinyl)-Sar were...... been suggested. However, these are not necessarily predictive of compounds that are actually translocated by hPEPT1. More information on affinity to and translocation via hPEPT1 of side-chain-modified dipeptides may be gained by conducting a study of selected dipeptide derivatives with variety in size...... translocated by hPEPT1. hPEPT1 is very susceptible to modifications of the N-terminal amino acid side chain of dipeptidomimetic substrates, in terms of achieving compounds with high affinity for the transporter. However, as affinity is not predictive of translocation, derivatization in this position must...

  2. The Cutting Edge of Affinity Electrophoresis Technology.

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-03-18

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years.

  3. Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

    Science.gov (United States)

    Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

    2017-05-01

    Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

  4. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    Science.gov (United States)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  5. Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

    Science.gov (United States)

    Tong, Jiefei; Cao, Biyin; Martyn, Gregory D; Krieger, Jonathan R; Taylor, Paul; Yates, Bradley; Sidhu, Sachdev S; Li, Shawn S C; Mao, Xinliang; Moran, Michael F

    2017-03-01

    Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. In vitro evolution and affinity-maturation with Coliphage qβ display.

    Directory of Open Access Journals (Sweden)

    Claudia Skamel

    Full Text Available The Escherichia coli bacteriophage, Qβ (Coliphage Qβ, offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV. DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.

  7. Biological applications of zinc imidazole framework through protein encapsulation

    Directory of Open Access Journals (Sweden)

    Pawan Kumar

    2015-12-01

    Full Text Available Abstract The robustness of biomolecules is always a significant challenge in the application of biostorage in biotechnology or pharmaceutical research. To learn more about biostorage in porous materials, we investigated the feasibility of using zeolite imidazolate framework (ZIF-8 with respect to protein encapsulation. Here, bovine serum albumin (BSA was selected as a model protein for encapsulation with the synthesis of ZIF-8 using water as a media. ZIF-8 exhibited excellent protein adsorption capacity through successive adsorption of free BSA with the formation of hollow crystals. The loading of protein in ZIF-8 crystals is affected by the molecular weight due to diffusion-limited permeation inside the crystals and also by the affinity of the protein to the pendent group on the ZIF-8 surface. The polar nature of BSA not only supported adsorption on the solid surface, but also enhanced the affinity of crystal spheres through weak coordination interactions with the ZIF-8 framework. The novel approach tested in this study was therefore successful in achieving protein encapsulation with porous, biocompatible, and decomposable microcrystalline ZIF-8. The presence of both BSA and FITC–BSA in ZIF-8 was confirmed consistently by spectroscopy as well as optical and electron microscopy.

  8. Proteins feel more than they see : Fine-tuning of binding affinity by properties of the non-interacting surface

    NARCIS (Netherlands)

    Kastritis, Panagiotis L.|info:eu-repo/dai/nl/315886668; Garcia Lopes Maia Rodrigues, João|info:eu-repo/dai/nl/330827391; Folkers, Gert E.|info:eu-repo/dai/nl/162277202; Boelens, Rolf|info:eu-repo/dai/nl/070151407; Bonvin, Alexandre M J J|info:eu-repo/dai/nl/113691238

    2014-01-01

    Protein-protein complexes orchestrate most cellular processes such as transcription, signal transduction and apoptosis. The factors governing their affinity remain elusive however, especially when it comes to describing dissociation rates (koff). Here we demonstrate that, next to direct

  9. Understanding Ion Binding Affinity and Selectivity in β-Parvalbumin Using Molecular Dynamics and Mean Spherical Approximation Theory.

    Science.gov (United States)

    Kucharski, Amir N; Scott, Caitlin E; Davis, Jonathan P; Kekenes-Huskey, Peter M

    2016-08-25

    Parvalbumin (PV) is a globular calcium (Ca(2+))-selective protein expressed in a variety of biological tissues. Our computational studies of the rat β-parvalbumin (β-PV) isoform seek to elucidate the molecular thermodynamics of Ca(2+) versus magnesium (Mg(2+)) binding at the protein's two EF-hand motifs. Specifically, we have utilized molecular dynamics (MD) simulations and a mean-field electrolyte model (mean spherical approximation (MSA) theory) to delineate how the EF-hand scaffold controls the "local" thermodynamics of Ca(2+) binding selectivity over Mg(2+). Our MD simulations provide the probability density of metal-chelating oxygens within the EF-hand scaffolds for both Ca(2+) and Mg(2+), as well the conformational strain induced by Mg(2+) relative to Ca(2+) binding. MSA theory utilizes the binding domain oxygen and charge distributions to predict the chemical potential of ion binding, as well as their corresponding concentrations within the binding domain. We find that the electrostatic and steric contributions toward ion binding were similar for Mg(2+) and Ca(2+), yet the latter was 5.5 kcal/mol lower in enthalpy when internal strain within the EF hand was considered. We therefore speculate that beyond differences in dehydration energies for the Ca(2+) versus Mg(2+), strain induced in the β-PV EF hand by cation binding significantly contributes to the nearly 10,000-fold difference in binding affinity reported in the literature. We further complemented our analyses of local factors governing cation binding selectivity with whole-protein (global) contributions, such as interhelical residue-residue contacts and solvent exposure of hydrophobic surface. These contributions were found to be comparable for both Ca(2+)- and Mg(2+)-bound β-PV, which may implicate local factors, EF-hand strain, and dehydration, in providing the primary means of selectivity. We anticipate these methods could be used to estimate metal binding thermodynamics across a broad range of

  10. Comprehensive Transcriptome Analysis of Response to Nickel Stress in White Birch (Betula papyrifera.

    Directory of Open Access Journals (Sweden)

    Gabriel Theriault

    Full Text Available White birch (Betula papyrifera is a dominant tree species of the Boreal Forest. Recent studies have shown that it is fairly resistant to heavy metal contamination, specifically to nickel. Knowledge of regulation of genes associated with metal resistance in higher plants is very sketchy. Availability and annotation of the dwarf birch (B. nana enables the use of high throughout sequencing approaches to understanding responses to environmental challenges in other Betula species such as B. papyrifera. The main objectives of this study are to 1 develop and characterize the B. papyrifera transcriptome, 2 assess gene expression dynamics of B. papyrifera in response to nickel stress, and 3 describe gene function based on ontology. Nickel resistant and susceptible genotypes were selected and used for transcriptome analysis. A total of 208,058 trinity genes were identified and were assembled to 275,545 total trinity transcripts. The transcripts were mapped to protein sequences and based on best match; we annotated the B. papyrifera genes and assigned gene ontology. In total, 215,700 transcripts were annotated and were compared to the published B. nana genome. Overall, a genomic match for 61% transcripts with the reference genome was found. Expression profiles were generated and 62,587 genes were found to be significantly differentially expressed among the nickel resistant, susceptible, and untreated libraries. The main nickel resistance mechanism in B. papyrifera is a downregulation of genes associated with translation (in ribosome, binding, and transporter activities. Five candidate genes associated to nickel resistance were identified. They include Glutathione S-transferase, thioredoxin family protein, putative transmembrane protein and two Nramp transporters. These genes could be useful for genetic engineering of birch trees.

  11. The elution of certain protein affinity tags with millimolar concentrations of diclofenac.

    Science.gov (United States)

    Baliova, Martina; Juhasova, Anna; Jursky, Frantisek

    2015-12-01

    Diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid) is a sparingly soluble, nonsteroidal anti-inflammatory drug therapeutically acting at low micromolar concentrations. In pH range from 8 to 11, its aqueous solubility can be increased up to 200 times by the presence of counter ions such as sodium. Our protein interaction studies revealed that a millimolar concentration of sodium diclofenac is able to elute glutathione S-transferase (GST), cellulose binding protein (CBD), and maltose binding protein (MBP) but not histidine-tagged or PDZ-tagged proteins from their affinity resins. The elution efficiency of diclofenac is comparable with the eluting agents normally used at similar concentrations. Native gel electrophoresis of sodium diclofenac-treated proteins showed that the interaction is non-covalent and non-denaturing. These results suggest that sodium diclofenac, in addition to its pharmaceutical applications, can also be exploited as a lead for the development of new proteomics reagents. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. High Dietary Protein Intake and Protein-Related Acid Load on Bone Health.

    Science.gov (United States)

    Cao, Jay J

    2017-12-01

    Consumption of high-protein diets is increasingly popular due to the benefits of protein on preserving lean mass and controlling appetite and satiety. The paper is to review recent clinical research assessing dietary protein on calcium metabolism and bone health. Epidemiological studies show that long-term, high-protein intake is positively associated with bone mineral density and reduced risk of bone fracture incidence. Short-term interventional studies demonstrate that a high-protein diet does not negatively affect calcium homeostasis. Existing evidence supports that the negative effects of the acid load of protein on urinary calcium excretion are offset by the beneficial skeletal effects of high-protein intake. Future research should focus on the role and the degree of contribution of other dietary and physiological factors, such as intake of fruits and vegetables, in reducing the acid load and further enhancing the anabolic effects of protein on the musculoskeletal system.

  13. Nickel accumulation and storage in bradyrhizobium japonicum

    International Nuclear Information System (INIS)

    Maier, R.J.; Pihl, T.D.; Stults, L.; Sray, W.

    1990-01-01

    Hydrogenase-depressed (chemolithotrophic growth conditions) and heterotrophically grown cultures of Bradyrhizobium japonicum accumulated nickel about equally over a 3-h period. Both types of cultures accumulated nickel primarily in a form that was not exchangeable with NiCl 2 , and they accumulated much more Ni than would be needed for the Ni-containing hydrogenase. The nickel accumulated by heterotrophically incubated cultures could later be mobilized to allow active hydrogenase synthesis during derepression in the absence of nickel, while cells both grown with Ni and the derepressed without nickel had low hydrogenase activities. The level of activity in cells grown with Ni and then derepressed without nickel was about the same as that in cultures derepressed in the presence of nickel. The Ni accumulated by heterotrophically grown cultures was associated principally with soluble proteins rather than particulate material, and this Ni was not lost upon dialyzing an extract containing the soluble proteins against either Ni-containing or EDTA-containing buffer. However, this Ni was lost upon pronase or low pH treatments. The soluble Ni-binding proteins were partially purified by gel filtration and DEAE chromatography. They were not antigenically related to hydrogenase peptides. Much of the 63 Ni eluted as a single peak of 48 kilodaltons. Experiments involving immunuprecipitation of 63 Ni-containing hydrogenase suggested that the stored source of Ni in heterotrophic cultures that could later be mobilized into hydrogenase resided in the nonexchangeable Ni-containing fraction rather than in loosely bound or ionic forms

  14. Improving binding mode and binding affinity predictions of docking by ligand-based search of protein conformations: evaluation in D3R grand challenge 2015

    Science.gov (United States)

    Xu, Xianjin; Yan, Chengfei; Zou, Xiaoqin

    2017-08-01

    The growing number of protein-ligand complex structures, particularly the structures of proteins co-bound with different ligands, in the Protein Data Bank helps us tackle two major challenges in molecular docking studies: the protein flexibility and the scoring function. Here, we introduced a systematic strategy by using the information embedded in the known protein-ligand complex structures to improve both binding mode and binding affinity predictions. Specifically, a ligand similarity calculation method was employed to search a receptor structure with a bound ligand sharing high similarity with the query ligand for the docking use. The strategy was applied to the two datasets (HSP90 and MAP4K4) in recent D3R Grand Challenge 2015. In addition, for the HSP90 dataset, a system-specific scoring function (ITScore2_hsp90) was generated by recalibrating our statistical potential-based scoring function (ITScore2) using the known protein-ligand complex structures and the statistical mechanics-based iterative method. For the HSP90 dataset, better performances were achieved for both binding mode and binding affinity predictions comparing with the original ITScore2 and with ensemble docking. For the MAP4K4 dataset, although there were only eight known protein-ligand complex structures, our docking strategy achieved a comparable performance with ensemble docking. Our method for receptor conformational selection and iterative method for the development of system-specific statistical potential-based scoring functions can be easily applied to other protein targets that have a number of protein-ligand complex structures available to improve predictions on binding.

  15. Single-Step Affinity Purification for Fungal Proteomics ▿ †

    OpenAIRE

    Liu, Hui-Lin; Osmani, Aysha H.; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B.; De Souza, Colin P.; Osmani, Stephen A.

    2010-01-01

    A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

  16. Repeated patch testing to nickel during childhood do not induce nickel sensitization

    DEFF Research Database (Denmark)

    Søgaard Christiansen, Elisabeth

    2014-01-01

    Background: Previously, patch test reactivity to nickel sulphate in a cohort of unselected infants tested repeatedly at 3-72 months of age has been reported. A reproducible positive reaction at 12 and 18 months was selected as a sign of nickel sensitivity, provided a patch test with an empty Finn...

  17. Performance improvement of pasted nickel electrodes with multi-wall carbon nanotubes for rechargeable nickel batteries

    International Nuclear Information System (INIS)

    Song, Q.S.; Aravindaraj, G.K.; Sultana, H.; Chan, S.L.I.

    2007-01-01

    Carbon nanotubes (CNTs) were employed as a functional additive to improve the electrochemical performance of pasted nickel-foam electrodes for rechargeable nickel-based batteries. The nickel electrodes were prepared with spherical β-Ni(OH) 2 powder as the active material and various amounts of CNTs as additives. Galvanostatic charge/discharge cycling tests showed that in comparison with the electrode without CNTs, the pasted nickel electrode with added CNTs exhibited better electrochemical properties in the chargeability, specific discharge capacity, active material utilization, discharge voltage, high-rate capability and cycling stability. Meanwhile, the CNT addition also lowered the packing density of Ni(OH) 2 particles in the three-dimensional porous nickel-foam substrate, which could lead to the decrease in the active material loading and discharge capacity of the electrode. Hence, the amount of CNTs added to Ni(OH) 2 should be optimized to obtain a high-performance nickel electrode, and an optimum amount of CNT addition was found to be 3 wt.%. The superior electrochemical performance of the nickel electrode with CNTs could be attributed to lower electrochemical impedance and less γ-NiOOH formed during charge/discharge cycling, as indicated by electrochemical impedance spectroscopy and X-ray diffraction analyses. Thus, it was an effective method to improve the electrochemical properties of pasted nickel electrodes by adding an appropriate amount of CNTs to spherical Ni(OH) 2 as the active material

  18. [Construction and expression of fusion protein TRX-hJagged1 in E.coli BL21].

    Science.gov (United States)

    Li, Guo-Hui; Fan, Yu-Zhen; Huang, Si-Yong; Liu, Qiang; Yin, Dan-Dan; Liu, Li; Chen, Ren-An; Hao, Miao-Wang; Liang, Ying-Min

    2014-06-01

    This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

  19. Contaminated nickel scrap processing

    International Nuclear Information System (INIS)

    Compere, A.L.; Griffith, W.L.; Hayden, H.W.; Johnson, J.S. Jr.; Wilson, D.F.

    1994-12-01

    The DOE will soon choose between treating contaminated nickel scrap as a legacy waste and developing high-volume nickel decontamination processes. In addition to reducing the volume of legacy wastes, a decontamination process could make 200,000 tons of this strategic metal available for domestic use. Contaminants in DOE nickel scrap include 234 Th, 234 Pa, 137 Cs, 239 Pu (trace), 60 Co, U, 99 Tc, and 237 Np (trace). This report reviews several industrial-scale processes -- electrorefining, electrowinning, vapormetallurgy, and leaching -- used for the purification of nickel. Conventional nickel electrolysis processes are particularly attractive because they use side-stream purification of process solutions to improve the purity of nickel metal. Additionally, nickel purification by electrolysis is effective in a variety of electrolyte systems, including sulfate, chloride, and nitrate. Conventional electrorefining processes typically use a mixed electrolyte which includes sulfate, chloride, and borate. The use of an electrorefining or electrowinning system for scrap nickel recovery could be combined effectively with a variety of processes, including cementation, solvent extraction, ion exchange, complex-formation, and surface sorption, developed for uranium and transuranic purification. Selected processes were reviewed and evaluated for use in nickel side-stream purification. 80 refs

  20. The Cutting Edge of Affinity Electrophoresis Technology

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  1. Relationship between nickel allergy and diet

    Directory of Open Access Journals (Sweden)

    Sharma Ashimav

    2007-01-01

    Full Text Available Nickel is a ubiquitous trace element and it occurs in soil, water, air and of the biosphere. It is mostly used to manufacture stainless steel. Nickel is the commonest cause of metal allergy. Nickel allergy is a chronic and recurring skin problem; females are affected more commonly than males. Nickel allergy may develop at any age. Once developed, it tends to persist life-long. Nickel is present in most of the dietary items and food is considered to be a major source of nickel exposure for the general population. Nickel content in food may vary considerably from place to place due to the difference in nickel content of the soil. However, certain foods are routinely high in nickel content. Nickel in the diet of a nickel-sensitive person can provoke dermatitis. Careful selection of food with relatively low nickel concentration can bring a reduction in the total dietary intake of nickel per day. This can influence the outcome of the disease and can benefit the nickel sensitive patient.

  2. Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

    Directory of Open Access Journals (Sweden)

    O Rickard Nilsson

    Full Text Available Recent studies indicate that defective activity of complement factor H (FH is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

  3. Mechanisms of c-myc degradation by nickel compounds and hypoxia.

    Directory of Open Access Journals (Sweden)

    Qin Li

    2009-12-01

    Full Text Available Nickel (Ni compounds have been found to cause cancer in humans and animal models and to transform cells in culture. At least part of this effect is mediated by stabilization of hypoxia inducible factor (HIF1a and activating its downstream signaling. Recent studies reported that hypoxia signaling might either antagonize or enhance c-myc activity depending on cell context. We investigated the effect of nickel on c-myc levels, and demonstrated that nickel, hypoxia, and other hypoxia mimetics degraded c-myc protein in a number of cancer cells (A549, MCF-7, MDA-453, and BT-474. The degradation of the c-Myc protein was mediated by the 26S proteosome. Interestingly, knockdown of both HIF-1alpha and HIF-2alpha attenuated c-Myc degradation induced by Nickel and hypoxia, suggesting the functional HIF-1alpha and HIF-2alpha was required for c-myc degradation. Further studies revealed two potential pathways mediated nickel and hypoxia induced c-myc degradation. Phosphorylation of c-myc at T58 was significantly increased in cells exposed to nickel or hypoxia, leading to increased ubiquitination through Fbw7 ubiquitin ligase. In addition, nickel and hypoxia exposure decreased USP28, a c-myc de-ubiquitinating enzyme, contributing to a higher steady state level of c-myc ubiquitination and promoting c-myc degradation. Furthermore, the reduction of USP28 protein by hypoxia signaling is due to both protein degradation and transcriptional repression. Nickel and hypoxia exposure significantly increased the levels of dimethylated H3 lysine 9 at the USP28 promoter and repressed its expression. Our study demonstrated that Nickel and hypoxia exposure increased c-myc T58 phosphorylation and decreased USP28 protein levels in cancer cells, which both lead to enhanced c-myc ubiquitination and proteasomal degradation.

  4. The effect of multi-component adsorption on selectivity in ion exchange displacement systems.

    Science.gov (United States)

    Tugcu, N; Cramer, S M

    2005-01-21

    This paper examines chemically selective displacement chromatography using affinity ranking plots, batch displacer screening experiments, column displacements, multi-component adsorption isotherms and spectroscopy. The affinity ranking plot indicated that the displacers, sucrose octasulfate (SOS) and tatrazine, should possess sufficient affinity to displace the proteins amyloglucosidase and apoferritin over a wide range of operating conditions. In addition, the plots indicated that the separation of these proteins by displacement chromatography would be extremely difficult. Further, the two proteins were shown to have very similar retention times under shallow linear gradient conditions. When batch displacement experiments were carried out, both tartrazine and SOS exhibited significant selectivity differences with respect to their ability to displace these two proteins, in contrast to the affinity ranking plot results. Column displacement experiments carried out with sucrose octasulfate agreed with the predictions of the affinity ranking plots, with both proteins being displaced but poorly resolved under several column displacement conditions. On the other hand, column displacement with tartrazine as the displacer resulted in the selective displacement and partial purification of apoferritin. Single- and multi-component isotherms of the proteins with or without the presence of displacers were determined and were used to help explain the selectivity reversals observed in the column and batch displacement experiments. In addition, fluorescence and CD spectra suggested that the displacers did not induce any structural changes to either of the proteins. The results in this paper indicate that multi-component adsorption behavior can be exploited for creating chemically selective displacement separations.

  5. Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon Sulfolobus solfataricus to nickel challenge

    Directory of Open Access Journals (Sweden)

    Scaloni Andrea

    2007-08-01

    Full Text Available Abstract Background Exposure to nickel (Ni and its chemical derivatives has been associated with severe health effects in human. On the contrary, poor knowledge has been acquired on target physiological processes or molecular mechanisms of this metal in model organisms, including Bacteria and Archaea. In this study, we describe an analysis focused at identifying proteins involved in the recovery of the archaeon Sulfolobus solfataricus strain MT4 from Ni-induced stress. Results To this purpose, Sulfolobus solfataricus was grown in the presence of the highest nickel sulphate concentration still allowing cells to survive; crude extracts from treated and untreated cells were compared at the proteome level by using a bi-dimensional chromatography approach. We identified several proteins specifically repressed or induced as result of Ni treatment. Observed up-regulated proteins were largely endowed with the ability to trigger recovery from oxidative and osmotic stress in other biological systems. It is noteworthy that most of the proteins induced following Ni treatment perform similar functions and a few have eukaryal homologue counterparts. Conclusion These findings suggest a series of preferential gene expression pathways activated in adaptation response to metal challenge.

  6. Protein scaffolds for selective enrichment of metal ions

    Science.gov (United States)

    He, Chuan; Zhou, Lu; Bosscher, Michael

    2016-02-09

    Polypeptides comprising high affinity for the uranyl ion are provided. Methods for binding uranyl using such proteins are likewise provided and can be used, for example, in methods for uranium purification or removal.

  7. Pretreatments of porous silica for improving the activity of a nickel-loaded catalyst

    Energy Technology Data Exchange (ETDEWEB)

    Arai, M.; Ikushima, Y.; Nishiyama, Y.

    1986-02-01

    Nickel catalysts supported on porous silicas treated with water, ethanol, and 1-butanol were found to be more active than that supported on untreated silica for ethane hydrogenolysis and furan hydrogenation. To elucidate the reason for the activation, some surface properties of the treated silicas were examined. The treatments decreased the surface area and increased the volume of the macropores, the surface density of hydroxyl groups, and the heat of immersion in water per unit surface area. The state of nickel on the support was examined by temperature programed desorption (TPD) of hydrogen, X-ray diffraction (XRD), and scanning electron microscopy/X-ray microanalysis. The dispersion measured by XRD was not appreciably altered by the treatments, but the TPD showed more hydrogen desorbed above 150/sup 0/C from nickel on the treated supports. The activation of nickel by pretreatment of the support was ascribed to the increase of those nickel atoms which dispersed finely over the support by the increased number of surface hydroxyl groups. This was based on the number of hydroxyl groups per unit surface area. The nickel catalyst supported on the pretreated silicas showed greater thermal stability. 16 references, 5 figures, 1 table.

  8. Evaluation of chromium, nickel, iron and manganese content in wheat, flour, bran and selected baked products

    Directory of Open Access Journals (Sweden)

    Bawiec Piotr

    2014-06-01

    Full Text Available Considering the nutritional values, breadstuff plays a big part in covering human nourishment needs and constitutes a base of all day diet. Moreover, bread is an excellent source of numerous vitamins and minerals the abundance of which depends on the degree of grinding. Thus, it seems to be very important to know the composition and level of bio-elements. That is why the main target of this study was to evaluate the concentration of selected trace elements: chromium (Cr, nickel (Ni, iron (Fe and manganese (Mn in wheat grain, wheat bran, different wheat and rye flour types and variety of breadstuff also with addition of grains and seeds from different bakeries and mills. Another task was to analyze if the technological process has an influence on secondary despoil of bread goods with heavy metal elements. The analyzed trace elements were measured with a precise and accurate atomic absorption spectrophotometric method (AAS and the results were expressed in mg/kg of selected sample. Obtained results show that bread and grain products are a good source of trace elements like chromium, nickel, iron and manganese. However, the higher levels of chromium and nickel in bread goods could rather be an effect of impurity caused by a technological process in mill and bakeries.

  9. Expression Changes in Metal-Resistance Genes in Microbacterium liquefaciens Under Nickel and Vanadium Exposure.

    Science.gov (United States)

    Fierros-Romero, Grisel; Wrosek-Cabrera, José A; Gómez-Ramírez, Marlenne; Pless, Reynaldo C; Rivas-Castillo, A M; Rojas-Avelizapa, Norma G

    2017-07-01

    Microbacterium liquefaciens MNSH2-PHGII-2 is a nickel-vanadium-resistant bacterium isolated from mine tailings located in Guanajuato, Mexico. In PHGII liquid media, M. liquefaciens has the ability to remove 29.5 ppm of Ni and 168.3 ppm of V. The present study reports, for the first time in M. liquefaciens, the presence of the genes nccA (Ni-Co-Cd resistance), hant (high-affinity nickel transporter), smtA, a metal-binding protein gene, and VAN2 (V resistance), which showed an increased expression under exposure to 200 ppm of Ni and 200 ppm of V during the logarithmic growth phase of the microorganism in PHGII liquid media. Data about the expression profile of genes conferring metal resistance to M. liquefaciens can improve the knowledge of those mechanisms involved in the processes of Ni-V resistance and probably in Ni-V removal processes. Based on our data, we can suggest that M. liquefaciens has the potential to be used in the biological treatment of toxic wastes with high Ni and V content.

  10. Contaminated nickel scrap processing

    Energy Technology Data Exchange (ETDEWEB)

    Compere, A.L.; Griffith, W.L.; Hayden, H.W.; Johnson, J.S. Jr.; Wilson, D.F.

    1994-12-01

    The DOE will soon choose between treating contaminated nickel scrap as a legacy waste and developing high-volume nickel decontamination processes. In addition to reducing the volume of legacy wastes, a decontamination process could make 200,000 tons of this strategic metal available for domestic use. Contaminants in DOE nickel scrap include {sup 234}Th, {sup 234}Pa, {sup 137}Cs, {sup 239}Pu (trace), {sup 60}Co, U, {sup 99}Tc, and {sup 237}Np (trace). This report reviews several industrial-scale processes -- electrorefining, electrowinning, vapormetallurgy, and leaching -- used for the purification of nickel. Conventional nickel electrolysis processes are particularly attractive because they use side-stream purification of process solutions to improve the purity of nickel metal. Additionally, nickel purification by electrolysis is effective in a variety of electrolyte systems, including sulfate, chloride, and nitrate. Conventional electrorefining processes typically use a mixed electrolyte which includes sulfate, chloride, and borate. The use of an electrorefining or electrowinning system for scrap nickel recovery could be combined effectively with a variety of processes, including cementation, solvent extraction, ion exchange, complex-formation, and surface sorption, developed for uranium and transuranic purification. Selected processes were reviewed and evaluated for use in nickel side-stream purification. 80 refs.

  11. Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs.

    Science.gov (United States)

    Soheilypour, M; Mofrad, M R K

    2016-11-02

    Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus.

  12. Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

    Directory of Open Access Journals (Sweden)

    Guy Caljon

    Full Text Available Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2 display DNA/RNA non-specific, high affinity nucleic acid binding with K(D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

  13. High-throughput bioscreening system utilizing high-performance affinity magnetic carriers exhibiting minimal non-specific protein binding

    International Nuclear Information System (INIS)

    Hanyu, Naohiro; Nishio, Kosuke; Hatakeyama, Mamoru; Yasuno, Hiroshi; Tanaka, Toshiyuki; Tada, Masaru; Nakagawa, Takashi; Sandhu, Adarsh; Abe, Masanori; Handa, Hiroshi

    2009-01-01

    For affinity purification of drug target protein we have developed magnetic carriers, narrow in size distribution (184±9 nm), which exhibit minimal non-specific binding of unwanted proteins. The carriers were highly dispersed in aqueous solutions and highly resistant to organic solvents, which enabled immobilization of various hydrophobic chemicals as probes on the carrier surfaces. Utilizing the carriers we have automated the process of separation and purification of the target proteins that had been done by manual operation previously.

  14. Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

    Science.gov (United States)

    Meiby, Elinor; Knapp, Stefan; Elkins, Jonathan M; Ohlson, Sten

    2012-11-01

    Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.

  15. Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag.

    Science.gov (United States)

    Li, Junhua; Zhang, Yang; Yang, Yanjun

    2013-03-01

    The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91-95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023-1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203-273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1-60, 203-273) and L2 (203-273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203-273) fusion protein on diatomite was shorter than that of L2 (1-60, 203-273) fusion protein. The maximum adsorption capacity of L2 (203-273) fusion protein was larger than that of L2 (1-60, 203-273) fusion protein. In order to study whether the L2 (203-273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203-273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203-273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme.

  16. Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.

    Science.gov (United States)

    Finzi, Andrés; Cloutier, Jonathan; Cohen, Eric A

    2003-07-01

    The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

  17. The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-C{beta} antibody

    Energy Technology Data Exchange (ETDEWEB)

    Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Jin, Aishun [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Department of Immunology, College of Basic Medical Sciences, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin 150081 (China); Kishi, Hiroyuki, E-mail: immkishi@med.u-toyama.ac.jp [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Muraguchi, Atsushi [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V

  18. Application of coupled affinity-sizing chromatography for the detection of proteolyzed HSA-tagged proteins.

    Science.gov (United States)

    London, Anne Serdakowski; Patel, Kunal; Quinn, Lisa; Lemmerer, Martin

    2015-04-01

    Coupled affinity liquid chromatography and size exclusion chromatography (ALC-SEC) is a technique that has been shown to successfully report product quality of proteins during cell expression and prior to the commencement of downstream processing chromatography steps. This method was applied to monitoring the degradation and subsequent partial remediation of a HSA-tagged protein which showed proteolysis, allowing for rapid cell line development to address this product quality dilemma. This paper outlines the novel application of this method for measuring and addressing protease-induced proteolysis. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.

    Science.gov (United States)

    Mulero, Maria Carmen; Shahabi, Shandy; Ko, Myung Soo; Schiffer, Jamie M; Huang, De-Bin; Wang, Vivien Ya-Fan; Amaro, Rommie E; Huxford, Tom; Ghosh, Gourisankar

    2018-05-22

    Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.

  20. Compound immobilization and drug-affinity chromatography.

    Science.gov (United States)

    Rix, Uwe; Gridling, Manuela; Superti-Furga, Giulio

    2012-01-01

    Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatography step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry.

  1. Ligand-Induced Cross-Linking of Z-Elastin-like Polypeptide-Functionalized E2 Protein Nanoparticles for Enhanced Affinity Precipitation of Antibodies.

    Science.gov (United States)

    Swartz, Andrew R; Sun, Qing; Chen, Wilfred

    2017-05-08

    Affinity precipitation is an ideal alternative to chromatography for antibody purification because it combines the high selectivity of an affinity ligand with the operational benefits of precipitation. However, the widespread use of elastin-like polypeptide (ELP) capture scaffolds for antibody purification has been hindered by the high salt concentrations and temperatures necessary for efficient ELP aggregation. In this paper, we employed a tandem approach to enhance ELP aggregation by enlarging the dimension of the capturing scaffold and by creating IgG-triggered scaffold cross-linking. This was accomplished by covalently conjugating the Z-domain-ELP (Z-ELP) capturing scaffold to a 25 nm diameter E2 protein nanocage using Sortase A ligation. We demonstrated the isothermal recovery of IgG in the virtual absence of salt due to the significantly increased scaffold dimension and cross-linking from multivalent IgG-E2 interactions. Because IgG cross-linking is reversible at low pH, it may be feasible to achieve a high yielding IgG purification by isothermal phase separation using a simple pH trigger.

  2. Kinetics of nickel bioaccumulation and its relevance to selected cellular processes in leaves of Elodea canadensis during short-term exposure.

    Science.gov (United States)

    Maleva, Maria G; Malec, Przemysław; Prasad, Majeti Narasimha Vara; Strzałka, Kazimierz

    2016-03-01

    Elodea canadensis is an aquatic macrophyte used widely as a bioindicator for the monitoring of water quality and in the phytoremediation of metal-contaminated waters. This study considers the kinetics of nickel bioaccumulation and changes in accompanying metabolic and stress-related physiological parameters. These include photosynthetic activity, pigment content, the accumulation of thiol-containing compounds, thiobarbituric acid-reactive substance (TBARS) products, and the activity of selected antioxidant enzymes (catalase, glutathione reductase, superoxide dismutase). Elodea leaves accumulated nickel according to pseudo-second-order kinetics, and the protective responses followed a time sequence which was related to the apparent rates of nickel accumulation. The applicability of second-order kinetics to the Ni uptake by Elodea leaves during the first 8 h of exposure to the metal suggested that the passive binding of metal ions (chemisorption) was a rate-limiting step at the initial phase of Ni accumulation. This phase was accompanied by an increase in photosynthetic activity together with elevated photosynthetic pigments and protein synthesis, the enhanced activity of antioxidant enzymes, and increased thiol concentration. In contrast, there was a decrease in metabolic activity upon the accumulation of TBARS, and the decline in enzyme activity was observed in the saturation phase of Ni accumulation (8-24 h). These results show that a correlation exists between the protective response and the apparent kinetic rate of Ni uptake. Thus, the time of exposure to the toxicant is a crucial factor in the activation of specific mechanisms of Ni detoxification and stress alleviation.

  3. A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

    Science.gov (United States)

    Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

    2018-04-30

    Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Selective induction of high-ouabain-affinity isoform of Na+-K+-ATPase by thyroid hormone

    International Nuclear Information System (INIS)

    Haber, R.S.; Loeb, J.N.

    1988-01-01

    The administration of thyroid hormone is known to result in an induction of the Na + -K + -adenosinetriphosphatase (Na + -K + -ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissues. Since the Na + -K + -ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain. The authors have studied the effects of 3,3',5-triiodo-L-thyronine (T 3 ) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na + -K + -ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (K I s) for ouabain of ∼10 -7 and 10 -4 M, respectively. Measurement of the specific binding of [ 3 H]ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (K d ) of slightly less than 10 -7 M, whose maximal binding capacity was increased by T 3 treatment by 185%. Binding studies in unfractionated homogenates of diaphragm similarly demonstrated the presence of high-affinity sites whose maximal binding capacity was increased by T 3 treatment. Quantitation of both the high- and low-ouabain-affinity forms of the Na + -K + -ATPase by ouabain-dependent phosphorylation from [ 32 P]orthophosphate confirmed that T 3 treatment markedly increased the number of high-affinity sites while having little effect on the number of low-affinity sites. These observations provide, to our knowledge, the first demonstration that these two forms of the Na + -K + -ATPase are subject to selective hormonal induction

  5. Nickel release from inexpensive jewelry and hair clasps purchased in an EU country - Are consumers sufficiently protected from nickel exposure?

    DEFF Research Database (Denmark)

    Thyssen, Jacob Pontoppidan; Menné, Torkil; Johansen, Jeanne Duus

    2009-01-01

    BACKGROUND: Nickel allergic subjects are at risk factor of acquiring hand eczema. In 1990 and 1994, respectively, Denmark and member states in the EU regulated nickel release from selected consumer products. The intention was that the nickel epidemic could be controlled and prevented if the general...... population was protected from high cutaneous nickel concentrations. Despite a decrease, the prevalence of nickel allergy remains high as nearly 10% of young women are nickel allergic. OBJECTIVE: This study aimed to perform dimethylglyoxime (DMG) testing of inexpensive jewelry and hair clasps purchased from...

  6. Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W

    2005-01-01

    To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting...... that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization...... of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level....

  7. Chemoorganotrophic Bioleaching of Olivine for Nickel Recovery

    Directory of Open Access Journals (Sweden)

    Yi Wai Chiang

    2014-06-01

    Full Text Available Bioleaching of olivine, a natural nickel-containing magnesium-iron-silicate, was conducted by applying chemoorganotrophic bacteria and fungi. The tested fungus, Aspergillus niger, leached substantially more nickel from olivine than the tested bacterium, Paenibacillus mucilaginosus. Aspergillus niger also outperformed two other fungal species: Humicola grisae and Penicillium chrysogenum. Contrary to traditional acid leaching, the microorganisms leached nickel preferentially over magnesium and iron. An average selectivity factor of 2.2 was achieved for nickel compared to iron. The impact of ultrasonic conditioning on bioleaching was also tested, and it was found to substantially increase nickel extraction by A. niger. This is credited to an enhancement in the fungal growth rate, to the promotion of particle degradation, and to the detachment of the stagnant biofilm around the particles. Furthermore, ultrasonic conditioning enhanced the selectivity of A. niger for nickel over iron to a value of 3.5. Pre-carbonating the olivine mineral, to enhance mineral liberation and change metal speciation, was also attempted, but did not result in improvement as a consequence of the mild pH of chemoorganotrophic bioleaching.

  8. Rational design of peptide affinity ligands for the purification of therapeutic enzymes.

    Science.gov (United States)

    Trasatti, John P; Woo, James; Ladiwala, Asif; Cramer, Steven; Karande, Pankaj

    2018-04-25

    Non-mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof-of-concept for developing affinity peptide-based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

  9. Visual short-term memory load strengthens selective attention.

    Science.gov (United States)

    Roper, Zachary J J; Vecera, Shaun P

    2014-04-01

    Perceptual load theory accounts for many attentional phenomena; however, its mechanism remains elusive because it invokes underspecified attentional resources. Recent dual-task evidence has revealed that a concurrent visual short-term memory (VSTM) load slows visual search and reduces contrast sensitivity, but it is unknown whether a VSTM load also constricts attention in a canonical perceptual load task. If attentional selection draws upon VSTM resources, then distraction effects-which measure attentional "spill-over"-will be reduced as competition for resources increases. Observers performed a low perceptual load flanker task during the delay period of a VSTM change detection task. We observed a reduction of the flanker effect in the perceptual load task as a function of increasing concurrent VSTM load. These findings were not due to perceptual-level interactions between the physical displays of the two tasks. Our findings suggest that perceptual representations of distractor stimuli compete with the maintenance of visual representations held in memory. We conclude that access to VSTM determines the degree of attentional selectivity; when VSTM is not completely taxed, it is more likely for task-irrelevant items to be consolidated and, consequently, affect responses. The "resources" hypothesized by load theory are at least partly mnemonic in nature, due to the strong correspondence they share with VSTM capacity.

  10. Liquid membrane ion-selective electrodes for potentiometric dosage of coper and nickel

    Directory of Open Access Journals (Sweden)

    MARIA PLENICEANY

    2005-02-01

    Full Text Available This paper presents experimental and theoretical data regarding the preparation and characterization of three liquid-membrane electrodes, which have not been mentioned in the specialized literature so far. The active substances, the solutions of which in nitrobenzene formed the membranes on a graphite rod, are simple complex combinations of Cu(II and Ni(II ions with an organic ligand belonging to the Schiff base class: N-[2-thienylmethilidene]-2-aminoethanol (TNAHE. The Cu2+ -selective and Ni2+ -selective electrodes were used to determine the copper and nickel ions in aqueous solutions, both by direct potentiometry and by potentiometric titration with EDTA. They were also used for the determination of Cu2+ and Ni2+ ions in industrial waters by direct potentiometry.

  11. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

    International Nuclear Information System (INIS)

    Bayne, M.L.; Applebaum, J.; Chicchi, G.G.; Hayes, N.S.; Green, B.G.; Cascieri, M.A.

    1988-01-01

    Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe -1 , Val 1 , Asn 2 , Gln 3 , His 4 , Ser 8 , His 9 , Glu 12 , Tyr 15 , Leu 16 ]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln 3 , Ala 4 ] IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr 15 , Leu 16 ] IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, [Gln 3 , Ala 4 , Tyr 15 ,Leu 16 ]IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I

  12. Nickel Inhibits Mitochondrial Fatty Acid Oxidation

    Science.gov (United States)

    Uppala, Radha; McKinney, Richard W.; Brant, Kelly A.; Fabisiak, James P.; Goetzman, Eric S.

    2015-01-01

    Nickel exposure is associated with changes in cellular energy metabolism which may contribute to its carcinogenic properties. Here, we demonstrate that nickel strongly represses mitochondrial fatty acid oxidation—the pathway by which fatty acids are catabolized for energy—in both primary human lung fibroblasts and mouse embryonic fibroblasts. At the concentrations used, nickel suppresses fatty acid oxidation without globally suppressing mitochondrial function as evidenced by increased glucose oxidation to CO2. Pre-treatment with L-carnitine, previously shown to prevent nickel-induced mitochondrial dysfunction in neuroblastoma cells, did not prevent the inhibition of fatty acid oxidation. The effect of nickel on fatty acid oxidation occurred only with prolonged exposure (>5 hr), suggesting that direct inhibition of the active sites of metabolic enzymes is not the mechanism of action. Nickel is a known hypoxia-mimetic that activates hypoxia inducible factor-1α (HIF1α). Nickel-induced inhibition of fatty acid oxidation was blunted in HIF1α knockout fibroblasts, implicating HIF1α as one contributor to the mechanism. Additionally, nickel down-regulated the protein levels of the key fatty acid oxidation enzyme very long-chain acyl-CoA dehydrogenase (VLCAD) in a dose-dependent fashion. In conclusion, inhibition of fatty acid oxidation by nickel, concurrent with increased glucose metabolism, represents a form of metabolic reprogramming that may contribute to nickel-induced carcinogenesis. PMID:26051273

  13. Non-affine deformation in microstructure selection in solids II: Elastoplastic theory for the dynamics of solid state transformations

    Energy Technology Data Exchange (ETDEWEB)

    Paul, Arya; Bhattacharya, Jayee; Sengupta, Surajit [S N Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Calcutta 700 098 (India); Rao, Madan [Raman Research Institute, C V Raman Avenue, Bangalore 560 080 (India)

    2008-09-10

    We study the nucleation dynamics of a model solid state transformation and the criterion for microstructure selection. Using a molecular dynamics (MD) simulation, we had shown that the dynamics of the solid is accompanied by the creation of transient non-affine zones (NAZ), which evolve with the rapidly moving transformation front. Guided by our MD results, we formulate a dynamical continuum theory of solid state transformation, which couples the elastic strain to the non-affine deformation. We demonstrate that our elastoplastic description recovers all qualitative features of the MD simulation. We construct a dynamical phase diagram for microstructure selection, including regimes where martensite or ferrite obtains, in addition to making several testable predictions.

  14. Magnetic Microbead Affinity Selection Screening (MagMass) of Botanical Extracts for Inhibitors of 15-Lipoxygenase

    Science.gov (United States)

    Rush, Michael D.; Walker, Elisabeth M.; Burton, Tristesse; van Breemen, Richard B.

    2016-01-01

    To expedite the identification of active natural products in complex mixtures such as botanical extracts, a Magnetic Microbead Affinity Selection Screening (MagMASS) procedure was developed. This technique utilizes target proteins immobilized on magnetic beads for rapid bioaffinity isolation of ligands from complex mixtures. A MagMASS method was developed and validated for 15-lipoxygenase. As a proof of concept, several North American prairie plants used medicinally by Native Americans were extracted with MeOH and screened. A hit from an extract of Proserpinaca palustris, also known as mermaid weed, was flagged for further characterization using high-resolution tandem mass spectrometry, dereplication, and identification using XCMS online. Through the application of high-resolution product ion tandem mass spectrometry, comparison with natural product databases and confirmation using standards, the hit was identified as quercitrin, which is a known inhibitor of 15-lipoxygenase. The overall workflow of MagMASS is faster and more amendable to automation than alternative methods designed for screening botanical extracts or complex mixtures of combinatorial libraries. PMID:27802026

  15. Characterizing complex polysera produced by antigen-specific immunization through the use of affinity-selected mimotopes.

    Directory of Open Access Journals (Sweden)

    Galina Denisova

    Full Text Available BACKGROUND: Antigen-based (as opposed to whole organism vaccines are actively being pursued for numerous indications. Even though different formulations may produce similar levels of total antigen-specific antibody, the composition of the antibody response can be quite distinct resulting in different levels of therapeutic activity. METHODOLOGY/PRINCIPAL FINDINGS: Using plasmid-based immunization against the proto-oncogene HER-2 as a model, we have demonstrated that affinity-selected epitope mimetics (mimotopes can provide a defined signature of a polyclonal antibody response. Further, using novel computer algorithms that we have developed, these mimotopes can be used to predict epitope targets. CONCLUSIONS/SIGNIFICANCE: By combining our novel strategy with existing methods of epitope prediction based on physical properties of an individual protein, we believe that this method offers a robust method for characterizing the breadth of epitope-specificity within a specific polyserum. This strategy is useful as a tool for monitoring immunity following vaccination and can also be used to define relevant epitopes for the creation of novel vaccines.

  16. A novel venom protein of the Asian bee (Apis cerana indica with an affinity to human α1-microglobulin

    Directory of Open Access Journals (Sweden)

    Rosdiana Natzir

    1999-01-01

    Full Text Available Bee stings are a common health problem throughout the world and can sometimes result in fatal anaphylactic reactions. We have studied Asian bee (Apis cerana indica, Apis cerana nigrocincta and Apis dorsata venoms and have discovered a novel protein with a molecular size of 50 kDa (p50, as shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, which has not been reported in the venom of the Western honey-bee, Apis mellifera (AM. The p50 protein showed a unique affinity to human α1-microglobulin (α1-m. As a result, p50 was purified using an affinity column with α1-m. The p50 protein was further purified by an affinity column with a monoclonal antibody raised against p50 in mice. The p50 protein induced an inflammatory reaction following injection into mouse ear; that is, degranulation of mast cells, edema, hyperemia and hyperpermeation of the local capillaries were observed. The reaction was very similar to that seen when phospholipase A2 of AM, a representative bee venom, was administered by injection. The inflammatory reaction induced by p50 was completely inhibited by mixing p50 with α1-m prior to injection. These results indicate that p50 is a unique venom component of the Asian bee that induces the inflammatory reaction and that human α1-m may be involved as a protective mechanism against bee stings of at least some Asian bee species.

  17. Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

    Energy Technology Data Exchange (ETDEWEB)

    James, W.M.; Emerick, M.C.; Agnew, W.S. (Yale Univ. School of medicine, New Haven, CT (USA))

    1989-07-11

    The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis {alpha}-(2{yields}8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3,200 pmol of ({sup 3}H)TTX-binding sites/mg of protein and a single polypeptide of {approximately}285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

  18. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

    Directory of Open Access Journals (Sweden)

    Samantha L Eaton

    Full Text Available Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate

  19. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    Science.gov (United States)

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  20. Perceptual Load Influences Selective Attention across Development

    Science.gov (United States)

    Couperus, Jane W.

    2011-01-01

    Research suggests that visual selective attention develops across childhood. However, there is relatively little understanding of the neurological changes that accompany this development, particularly in the context of adult theories of selective attention, such as N. Lavie's (1995) perceptual load theory of attention. This study examined visual…

  1. Differential gene expression profile of the calanoid copepod, Pseudodiaptomus annandalei, in response to nickel exposure.

    Science.gov (United States)

    Jiang, Jie-Lan; Wang, Gui-Zhong; Mao, Ming-Guang; Wang, Ke-Jian; Li, Shao-Jing; Zeng, Chao-Shu

    2013-03-01

    To better understand the underlying mechanisms of reactions of copepods exposed to elevated level of nickel, the suppression subtractive hybridization (SSH) was used to elucidate the response of the copepod Pseudodiaptomus annandalei to nickel exposure at the gene level. P. annandale is one of a few copepod species that can be cultured relatively easy under laboratory condition, and it is considered to be a potential model species for toxicity study. In the present study, P. annandalei were exposed to nickel at a concentration of 8.86 mgL(-1) for 24h, after which the RNA was prepared for SSH using unexposed P. annandalei as drivers. A total of 474 clones on the middle scale in the SSH library were sequenced. Among these genes, 129 potential functional genes were recognized based on the BLAST searches in NCBI and Uniprot databases. These genes were then categorized into nine groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 129 genes, 127 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains (CD) and proteins using the CD-Search service and BLASTp. Among 129 genes, 119 (92.2%) were annotated to be involved in different biological processes, while 10 genes (7.8%) were classified as an unknown-function gene group. To further confirm the up-regulation of differentially expressed genes, the quantitative real time PCR were performed to test eight randomly selected genes, in which five of them, i.e. α-tubulin, ribosomal protein L13, ferritin, separase and Myohemerythrin-1, exhibited clear up-regulation after nickel exposure. In addition, MnSOD was further studied for the differential expression pattern after nickel exposure and the results showed that MnSOD had a time- and dose-dependent expression pattern in the copepod after nickel exposure. To the best of our knowledge, this is the first attempt to investigate the toxicity

  2. Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.

    Science.gov (United States)

    Karkov, Hanne Sophie; Krogh, Berit Olsen; Woo, James; Parimal, Siddharth; Ahmadian, Haleh; Cramer, Steven M

    2015-11-01

    In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules. © 2015 Wiley Periodicals, Inc.

  3. Nickel hydrogen bipolar battery electrode design

    Science.gov (United States)

    Puglisi, V. J.; Russell, P.; Verrier, D.; Hall, A.

    1985-01-01

    The preferred approach of the NASA development effort in nickel hydrogen battery design utilizes a bipolar plate stacking arrangement to obtain the required voltage-capacity configuration. In a bipolar stack, component designs must take into account not only the typical design considerations such as voltage, capacity and gas management, but also conductivity to the bipolar (i.e., intercell) plate. The nickel and hydrogen electrode development specifically relevant to bipolar cell operation is discussed. Nickel oxide electrodes, having variable type grids and in thicknesses up to .085 inch are being fabricated and characterized to provide a data base. A selection will be made based upon a system level tradeoff. Negative (hydrpogen) electrodes are being screened to select a high performance electrode which can function as a bipolar electrode. Present nickel hydrogen negative electrodes are not capable of conducting current through their cross-section. An electrode was tested which exhibits low charge and discharge polarization voltages and at the same time is conductive. Test data is presented.

  4. Aluminium fluoride and magnesium, activators of heterotrimeric GTP-binding proteins, affect high-affinity binding of the fungal toxin fusicoccin to the fusicoccin-binding protein in oat root plasma membranes.

    NARCIS (Netherlands)

    de Boer, A.H.; Van der Molen, G.W.; Prins, H.B.A.; Korthout, H.A.A.J.; van der Hoeven, P.C.J.

    1994-01-01

    The fusicoccin-binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high-affinity [H-3]fusicoccin binding to almost zero in a

  5. Urine nickel concentrations in nickel-exposed workers.

    Science.gov (United States)

    Bernacki, E J; Parsons, G E; Roy, B R; Mikac-Devic, M; Kennedy, C D; Sunderman, F W

    1978-01-01

    Electrothermal atomic absorption spectrometry was employed for analyses of nickel concentrations in urine samples from nickel-exposed workers in 10 occupational groups and from non-exposed workers in two control groups. Mean concentrations of nickel in urine were greatest in workers who were exposed to inhalation of aerosols of soluble nickel salts (e.g., workers in nickel plating operations and in an electrolytic nickel refinery). Less marked increases in urine nickel concentrations were found in groups of metal sprayers, nickel battery workers, bench mechanics and are welders. No significant increases in mean concentrations of nickel were found in urine samples from workers who performed grinding, buffing and polishing of nickel-containing alloys or workers in a coal gasification plant who employed Raney nickel as a hydrogenation catalyst. Measurements of nickel concentrations in urine are more sensitive and practical than measurements of serum nickel concentrations for evaluation of nickel exposures in industrial workers.

  6. Canadian House Dust Study: Population-based concentrations, loads and loading rates of arsenic, cadmium, chromium, copper, nickel, lead, and zinc inside urban homes

    Energy Technology Data Exchange (ETDEWEB)

    Rasmussen, Pat E. [Exposure and Biomonitoring Division, Healthy Environments and Consumer Safety Branch, Health Canada, 50 Colombine Driveway, Ottawa, ON, Canada K1A 0K9 (Canada); Department of Earth Sciences, University of Ottawa, 140 Louis Pasteur, Ottawa, ON, Canada K1N 6N5 (Canada); Levesque, Christine [Exposure and Biomonitoring Division, Healthy Environments and Consumer Safety Branch, Health Canada, 50 Colombine Driveway, Ottawa, ON, Canada K1A 0K9 (Canada); Chénier, Marc; Gardner, H. David [Exposure and Biomonitoring Division, Healthy Environments and Consumer Safety Branch, Health Canada, 50 Colombine Driveway, Ottawa, ON, Canada K1A 0K9 (Canada); Department of Earth Sciences, University of Ottawa, 140 Louis Pasteur, Ottawa, ON, Canada K1N 6N5 (Canada); Jones-Otazo, Heather [Regions and Programs Branch, Health Canada, 180 Queen Street West, Toronto, ON, Canada M5V 3L7 (Canada); Petrovic, Sanya [Contaminated Sites Division, Healthy Environments and Consumer Safety Branch, Health Canada, 269 Laurier Ave West, Ottawa, ON, Canada K1A 0K9 (Canada)

    2013-01-15

    The Canadian House Dust Study was designed to obtain nationally representative urban house dust metal concentrations (μg g{sup −1}) and metal loadings (μg m{sup −2}) for arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb), and zinc (Zn). Consistent sampling of active dust of known age and provenance (area sampled) also permitted the calculation of indoor loading rates (mg m{sup −2} day{sup −1} for dust and μg m{sup −2} day{sup −1} for metals) for the winter season (from 2007 to 2010) when houses are most tightly sealed. Geomean/median indoor dust loading rates in homes located more than 2 km away from industry of any kind (9.6/9.1 mg m{sup −2} day{sup −1}; n = 580) were significantly lower (p < .001) than geomean (median) dust loading rates in homes located within 2 km of industry (13.5/13.4 mg m{sup −2} day{sup −1}; n = 421). Proximity to industry was characterized by higher indoor metal loading rates (p < .003), but no difference in dust metal concentrations (.29 ≥ p ≤ .97). Comparisons of non-smokers' and smokers' homes in non-industrial zones showed higher metal loading rates (.005 ≥ p ≤ .038) in smokers' homes, but no difference in dust metal concentrations (.15 ≥ p ≤ .97). Relationships between house age and dust metal concentrations were significant for Pb, Cd and Zn (p < .001) but not for the other four metals (.14 ≥ p ≤ .87). All seven metals, however, displayed a significant increase in metal loading rates with house age (p < .001) due to the influence of higher dust loading rates in older homes (p < .001). Relationships between three measures of metals in house dust – concentration, load, and loading rate – in the context of house age, smoking behavior and urban setting consistently show that concentration data is a useful indicator of the presence of metal sources in the home, whereas dust mass is the overriding influence on metal loadings and loading rates

  7. Elicitin-induced distal systemic resistance in plants is mediated through the protein-protein interactions influenced by selected lysine residues

    Directory of Open Access Journals (Sweden)

    Hana eUhlíková

    2016-02-01

    Full Text Available Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium spp. classified as oomycete PAMPs. Although alfa- and beta-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, beta-elicitins (possessing 6-7 lysine residues are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the alfa-isoforms (with only 1-3 lysine residues.To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of beta-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein’s charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins’ movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance.

  8. Affinity maturation of a portable Fab–RNA module for chaperone-assisted RNA crystallography

    Science.gov (United States)

    Koirala, Deepak; Shelke, Sandip A; Dupont, Marcel; Ruiz, Stormy; DasGupta, Saurja; Bailey, Lucas J; Benner, Steven A; Piccirilli, Joseph A

    2018-01-01

    Abstract Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography. PMID:29309709

  9. Convergence of Domain Architecture, Structure, and Ligand Affinity in Animal and Plant RNA-Binding Proteins.

    Science.gov (United States)

    Dias, Raquel; Manny, Austin; Kolaczkowski, Oralia; Kolaczkowski, Bryan

    2017-06-01

    Reconstruction of ancestral protein sequences using phylogenetic methods is a powerful technique for directly examining the evolution of molecular function. Although ancestral sequence reconstruction (ASR) is itself very efficient, downstream functional, and structural studies necessary to characterize when and how changes in molecular function occurred are often costly and time-consuming, currently limiting ASR studies to examining a relatively small number of discrete functional shifts. As a result, we have very little direct information about how molecular function evolves across large protein families. Here we develop an approach combining ASR with structure and function prediction to efficiently examine the evolution of ligand affinity across a large family of double-stranded RNA binding proteins (DRBs) spanning animals and plants. We find that the characteristic domain architecture of DRBs-consisting of 2-3 tandem double-stranded RNA binding motifs (dsrms)-arose independently in early animal and plant lineages. The affinity with which individual dsrms bind double-stranded RNA appears to have increased and decreased often across both animal and plant phylogenies, primarily through convergent structural mechanisms involving RNA-contact residues within the β1-β2 loop and a small region of α2. These studies provide some of the first direct information about how protein function evolves across large gene families and suggest that changes in molecular function may occur often and unassociated with major phylogenetic events, such as gene or domain duplications. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  10. Distracted and confused?: selective attention under load.

    Science.gov (United States)

    Lavie, Nilli

    2005-02-01

    The ability to remain focused on goal-relevant stimuli in the presence of potentially interfering distractors is crucial for any coherent cognitive function. However, simply instructing people to ignore goal-irrelevant stimuli is not sufficient for preventing their processing. Recent research reveals that distractor processing depends critically on the level and type of load involved in the processing of goal-relevant information. Whereas high perceptual load can eliminate distractor processing, high load on "frontal" cognitive control processes increases distractor processing. These findings provide a resolution to the long-standing early and late selection debate within a load theory of attention that accommodates behavioural and neuroimaging data within a framework that integrates attention research with executive function.

  11. Genome-centric evaluation of Burkholderia sp. strain SRS-W-2-2016 resistant to high concentrations of uranium and nickel isolated from the Savannah River Site (SRS, USA

    Directory of Open Access Journals (Sweden)

    Ashish Pathak

    2017-06-01

    Full Text Available Savannah River Site (SRS, an approximately 800-km2 former nuclear weapons production facility located near Aiken, SC remains co-contaminated by heavy metals and radionuclides. To gain a better understanding on microbially-mediated bioremediation mechanisms, several bacterial strains resistant to high concentrations of Uranium (U and Nickel (Ni were isolated from the Steeds Pond soils located within the SRS site. One of the isolated strains, designated as strain SRS-W-2-2016, grew robustly on both U and Ni. To fully understand the arsenal of metabolic functions possessed by this strain, a draft whole genome sequence (WGS was obtained, assembled, annotated and analyzed. Genome-centric evaluation revealed the isolate to belong to the Burkholderia genus with close affiliation to B. xenovorans LB400, an aggressive polychlorinated biphenyl-degrader. At a coverage of 90×, the genome of strain SRS-W-2-2016 consisted of 8,035,584 bases with a total number of 7071 putative genes assembling into 191 contigs with an N50 contig length of 134,675 bases. Several gene homologues coding for resistance to heavy metals/radionuclides were identified in strain SRS-W-2-2016, such as a suite of outer membrane efflux pump proteins similar to nickel/cobalt transporter regulators, peptide/nickel transport substrate and ATP-binding proteins, permease proteins, and a high-affinity nickel-transport protein. Also noteworthy were two separate gene fragments in strain SRS-W-2-2016 homologous to the spoT gene; recently correlated with bacterial tolerance to U. Additionally, a plethora of oxygenase genes were also identified in the isolate, potentially involved in the breakdown of organic compounds facilitating the strain's successful colonization and survival in the SRS co-contaminated soils. The WGS project of Burkholderia sp. strain SRS-W-2-2016 is available at DDBJ/ENA/GenBank under the accession #MSDV00000000.

  12. Does airborne nickel exposure induce nickel sensitization?

    Science.gov (United States)

    Mann, Eugen; Ranft, Ulrich; Eberwein, Georg; Gladtke, Dieter; Sugiri, Dorothee; Behrendt, Heidrun; Ring, Johannes; Schäfer, Torsten; Begerow, Jutta; Wittsiepe, Jürgen; Krämer, Ursula; Wilhelm, Michael

    2010-06-01

    Nickel is one of the most prevalent causes of contact allergy in the general population. This study focuses on human exposure to airborne nickel and its potential to induce allergic sensitization. The study group consisted of 309 children at school-starter age living in the West of Germany in the vicinity of two industrial sources and in a rural town without nearby point sources of nickel. An exposure assessment of nickel in ambient air was available for children in the Ruhr district using routinely monitored ambient air quality data and dispersion modelling. Internal nickel exposure was assessed by nickel concentrations in morning urine samples of the children. The observed nickel sensitization prevalence rates varied between 12.6% and 30.7%. Statistically significant associations were showed between exposure to nickel in ambient air and urinary nickel concentration as well as between urinary nickel concentration and nickel sensitization. Furthermore, an elevated prevalence of nickel sensitization was associated with exposure to increased nickel concentrations in ambient air. The observed associations support the assumption that inhaled nickel in ambient air might be a risk factor for nickel sensitization; further studies in larger collectives are necessary.

  13. Protein-protein interaction site prediction in Homo sapiens and E. coli using an interaction-affinity based membership function in fuzzy SVM.

    Science.gov (United States)

    Sriwastava, Brijesh Kumar; Basu, Subhadip; Maulik, Ujjwal

    2015-10-01

    Protein-protein interaction (PPI) site prediction aids to ascertain the interface residues that participate in interaction processes. Fuzzy support vector machine (F-SVM) is proposed as an effective method to solve this problem, and we have shown that the performance of the classical SVM can be enhanced with the help of an interaction-affinity based fuzzy membership function. The performances of both SVM and F-SVM on the PPI databases of the Homo sapiens and E. coli organisms are evaluated and estimated the statistical significance of the developed method over classical SVM and other fuzzy membership-based SVM methods available in the literature. Our membership function uses the residue-level interaction affinity scores for each pair of positive and negative sequence fragments. The average AUC scores in the 10-fold cross-validation experiments are measured as 79.94% and 80.48% for the Homo sapiens and E. coli organisms respectively. On the independent test datasets, AUC scores are obtained as 76.59% and 80.17% respectively for the two organisms. In almost all cases, the developed F-SVM method improves the performances obtained by the corresponding classical SVM and the other classifiers, available in the literature.

  14. METHOD OF APPLYING NICKEL COATINGS ON URANIUM

    Science.gov (United States)

    Gray, A.G.

    1959-07-14

    A method is presented for protectively coating uranium which comprises etching the uranium in an aqueous etching solution containing chloride ions, electroplating a coating of nickel on the etched uranium and heating the nickel plated uranium by immersion thereof in a molten bath composed of a material selected from the group consisting of sodium chloride, potassium chloride, lithium chloride, and mixtures thereof, maintained at a temperature of between 700 and 800 deg C, for a time sufficient to alloy the nickel and uranium and form an integral protective coating of corrosion-resistant uranium-nickel alloy.

  15. Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr-activated sepharose-4B affinity chromatography.

    Science.gov (United States)

    Hedayati Ch, Mojtaba; Amani, Jafar; Sedighian, Hamid; Amin, Mohsen; Salimian, Jafar; Halabian, Raheleh; Imani Fooladi, Abbas Ali

    2016-09-01

    Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA-aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd  = 2.3 × 10(-11) ). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  16. Superior creep strength of a nickel-based superalloy produced by selective laser melting

    Energy Technology Data Exchange (ETDEWEB)

    Pröbstle, M., E-mail: martin.proebstle@fau.de [Department of Materials Science & Engineering Institute I, Friedrich-Alexander-Universität Erlangen-Nürnberg, Martensstraße 5, D-91058 Erlangen (Germany); Neumeier, S.; Hopfenmüller, J.; Freund, L.P. [Department of Materials Science & Engineering Institute I, Friedrich-Alexander-Universität Erlangen-Nürnberg, Martensstraße 5, D-91058 Erlangen (Germany); Niendorf, T. [Institut für Werkstofftechnik (Materials Engineering), Universität Kassel, Mönchebergstr. 3, D-34125 Kassel (Germany); Schwarze, D. [SLM Solutions GmbH, Roggenhorster Straße 9c, D-23556 Lübeck (Germany); Göken, M. [Department of Materials Science & Engineering Institute I, Friedrich-Alexander-Universität Erlangen-Nürnberg, Martensstraße 5, D-91058 Erlangen (Germany)

    2016-09-30

    The creep properties of a polycrystalline nickel-based superalloy produced via selective laser melting were investigated in this study. All heat treatment conditions of the additively manufactured material show superior creep strength compared to conventional cast and wrought material. The process leads to a microstructure with fine subgrains. In comparison to conventional wrought material no Niobium rich δ phase is necessary to control the grain size and thus more Niobium is available for precipitation hardening and solid solution strengthening resulting in improved creep strength.

  17. Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins : a wash step with a low concentration of EDTA

    NARCIS (Netherlands)

    Westra, DF; Welling, GW; Koedijk, DGAM; Scheffer, AJ; The, TH; Welling-Wester, S

    2001-01-01

    Immobilised metal-ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins in which a poly-histidine tag is introduced. However, other proteins may also bind to IMAC columns. We describe the use of a washing buffer with a low concentration of EDTA (0.5 mM) for

  18. Electroless nickel plating on abs plastics from nickel chloride and nickel sulfate baths

    International Nuclear Information System (INIS)

    Inam-ul-haque; Ahmad, S.; Khan, A.

    2005-01-01

    Aqueous acid nickel chloride and alkaline nickel sulphate bath were studied for electroless nickel planting on acrylonitrile-butadiene-styrene (ABS) plastic. Before electroless nickel plating, specimens were etched, sensitized and activated. Effects of sodium hypophosphite and sodium citrate concentration on the electroless nickel plating thickness were discussed. Aqueous acid nickel chloride bath comprising, nickel chloride 10 g/L, sodium hypophosphite 40 g/L, sodium citrate 40g/L at pH 5.5, temperature 85 deg. C and density of 1 Be/ for thirty minutes gave best coating thickness in micrometer. It was found that acid nickel chloride bath had a greater stability, wide operating range and better coating thickness results than alkaline nickel sulphate bath. Acid nickel chloride bath gave better coating thickness than alkaline nickel sulfate bath

  19. PepMapper: a collaborative web tool for mapping epitopes from affinity-selected peptides.

    Directory of Open Access Journals (Sweden)

    Wenhan Chen

    Full Text Available Epitope mapping from affinity-selected peptides has become popular in epitope prediction, and correspondingly many Web-based tools have been developed in recent years. However, the performance of these tools varies in different circumstances. To address this problem, we employed an ensemble approach to incorporate two popular Web tools, MimoPro and Pep-3D-Search, together for taking advantages offered by both methods so as to give users more options for their specific purposes of epitope-peptide mapping. The combined operation of Union finds as many associated peptides as possible from both methods, which increases sensitivity in finding potential epitopic regions on a given antigen surface. The combined operation of Intersection achieves to some extent the mutual verification by the two methods and hence increases the likelihood of locating the genuine epitopic region on a given antigen in relation to the interacting peptides. The Consistency between Intersection and Union is an indirect sufficient condition to assess the likelihood of successful peptide-epitope mapping. On average from 27 tests, the combined operations of PepMapper outperformed either MimoPro or Pep-3D-Search alone. Therefore, PepMapper is another multipurpose mapping tool for epitope prediction from affinity-selected peptides. The Web server can be freely accessed at: http://informatics.nenu.edu.cn/PepMapper/

  20. Mutational and Computational Evidence That a Nickel-Transfer Tunnel in UreD Is Used for Activation of Klebsiella aerogenes Urease.

    Science.gov (United States)

    Farrugia, Mark A; Wang, Beibei; Feig, Michael; Hausinger, Robert P

    2015-10-20

    Nickel-containing urease from Klebsiella aerogenes requires four accessory proteins for proper active site metalation. The metallochaperone UreE delivers nickel to UreG, a GTPase that forms a UreD/UreF/UreG complex, which binds to urease apoprotein via UreD. Prior in silico analysis of the homologous, structurally characterized UreH/UreF/UreG complex from Helicobacter pylori identified a water tunnel originating at a likely nickel-binding motif in UreG, passing through UreF, and exiting UreH, suggestive of a role for the channel in providing the metal to urease apoprotein for its activation; however, no experimental support was reported for the significance of this tunnel. Here, specific variants were designed to disrupt a comparable 34.6 Å predicted internal tunnel, alternative channels, and surface sites for UreD. Cells producing a set of tunnel-disrupting variants of UreD exhibited greatly reduced urease specific activities, whereas other mutants had no appreciable effect on activity. Affinity pull-down studies of cell-free extracts from tunnel-disrupting mutant cultures showed no loss of UreD interactions with urease or UreF/UreG. The nickel contents of urease samples enriched from activity-deficient cultures were decreased, while zinc and iron incorporation increased. Molecular dynamics simulations revealed size restrictions in the internal channels of the UreD variants. These findings support the role of a molecular tunnel in UreD as a direct facilitator of nickel transfer into urease, illustrating a new paradigm in active site metallocenter assembly.

  1. Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites.

    Science.gov (United States)

    Erspamer, V; Melchiorri, P; Falconieri-Erspamer, G; Negri, L; Corsi, R; Severini, C; Barra, D; Simmaco, M; Kreil, G

    1989-07-01

    Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.

  2. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    Science.gov (United States)

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  3. Nickel Dermatitis - Nickel Excretion

    DEFF Research Database (Denmark)

    Menné, T.; Thorboe, A.

    1976-01-01

    Nickel excretion in urine in four females -sensitive to nickel with an intermittent dyshidrotic eruption was measured with flameless atomic absorption. Excretion of nickel was found to be increased in association with outbreaks of vesicles. The results support the idea that the chronic condition ...

  4. Neural effects of cognitive control load on auditory selective attention.

    Science.gov (United States)

    Sabri, Merav; Humphries, Colin; Verber, Matthew; Liebenthal, Einat; Binder, Jeffrey R; Mangalathu, Jain; Desai, Anjali

    2014-08-01

    Whether and how working memory disrupts or alters auditory selective attention is unclear. We compared simultaneous event-related potentials (ERP) and functional magnetic resonance imaging (fMRI) responses associated with task-irrelevant sounds across high and low working memory load in a dichotic-listening paradigm. Participants performed n-back tasks (1-back, 2-back) in one ear (Attend ear) while ignoring task-irrelevant speech sounds in the other ear (Ignore ear). The effects of working memory load on selective attention were observed at 130-210ms, with higher load resulting in greater irrelevant syllable-related activation in localizer-defined regions in auditory cortex. The interaction between memory load and presence of irrelevant information revealed stronger activations primarily in frontal and parietal areas due to presence of irrelevant information in the higher memory load. Joint independent component analysis of ERP and fMRI data revealed that the ERP component in the N1 time-range is associated with activity in superior temporal gyrus and medial prefrontal cortex. These results demonstrate a dynamic relationship between working memory load and auditory selective attention, in agreement with the load model of attention and the idea of common neural resources for memory and attention. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Metal-conjugated affinity labels: A new concept to create enantioselective artificial metalloenzymes

    KAUST Repository

    Reiner, Thomas

    2013-02-20

    How to train a protein: Metal-conjugated affinity labels were used to selectively position catalytically active metal centers in the binding pocket of proteases. The resulting artificial metalloenzymes achieve up to 82% e.r. in the hydrogenation of ketones. The modular setup enables a rapid generation of artificial metalloenzyme libraries, which can be adapted to a broad range of catalytic conditions. 2013 The Authors.

  6. Metal-conjugated affinity labels: A new concept to create enantioselective artificial metalloenzymes

    KAUST Repository

    Reiner, Thomas; Jantke, Dominik; Marziale, Alexander N.; Raba, Andreas; Eppinger, Jö rg

    2013-01-01

    How to train a protein: Metal-conjugated affinity labels were used to selectively position catalytically active metal centers in the binding pocket of proteases. The resulting artificial metalloenzymes achieve up to 82% e.r. in the hydrogenation of ketones. The modular setup enables a rapid generation of artificial metalloenzyme libraries, which can be adapted to a broad range of catalytic conditions. 2013 The Authors.

  7. Proteomic Assessment of Biochemical Pathways That Are Critical to Nickel-Induced Toxicity Responses in Human Epithelial Cells

    Science.gov (United States)

    Ge, Yue; Bruno, Maribel; Haykal-Coates, Najwa; Wallace, Kathleen; Andrews, Debora; Swank, Adam; Winnik, Witold; Ross, Jeffrey A.

    2016-01-01

    Understanding the mechanisms underlying toxicity initiated by nickel, a ubiquitous environmental contaminant and known human carcinogen is necessary for proper assessment of its risks to human and environment. Among a variety of toxic mechanisms, disruption of protein responses and protein response-based biochemical pathways represents a key mechanism through which nickel induces cytotoxicity and carcinogenesis. To identify protein responses and biochemical pathways that are critical to nickel-induced toxicity responses, we measured cytotoxicity and changes in expression and phosphorylation status of 14 critical biochemical pathway regulators in human BEAS-2B cells exposed to four concentrations of nickel using an integrated proteomic approach. A subset of the pathway regulators, including interleukin-6, and JNK, were found to be linearly correlated with cell viability, and may function as molecular determinants of cytotoxic responses of BEAS-2B cells to nickel exposures. In addition, 128 differentially expressed proteins were identified by two dimensional electrophoresis (2-DE) and mass spectrometry. Principal component analysis, hierarchical cluster analyses, and ingenuity signaling pathway analysis (IPA) identified putative nickel toxicity pathways. Some of the proteins and pathways identified have not previously been linked to nickel toxicity. Based on the consistent results obtained from both ELISA and 2-DE proteomic analysis, we propose a core signaling pathway regulating cytotoxic responses of human BEAS-2B cells to nickel exposures, which integrates a small set of proteins involved in glycolysis and gluconeogenesis pathways, apoptosis, protein degradation, and stress responses including inflammation and oxidative stress. PMID:27626938

  8. Nickel brittling by hydrogen. Temperature effect

    International Nuclear Information System (INIS)

    Lapitz, P.A; Fernandez, S; Alvarez, M.G

    2006-01-01

    The results of a study on the effect of different variables on the susceptibility to brittling by hydrogen and the velocity of propagation of fissures in nickel wire (99.7% purity) are described. The hydrogen load was carried out by cathodic polarization in H 2 SO 4 0.5m solution. The susceptibility to brittling by hydrogen was determined with traction tests at slow deformation speed and constant cathodic potential, and the later observation of the fracture surface by scanning electron microscopy. The variables studied were: applied cathodic overpower, speed of initial deformation and temperature. The results showed that the speed of fissure propagation in the nickel by brittleness from hydrogen is a function of the applied potential and the speed of deformation used. Without tension, the hydrogen load by cathodic polarization at room temperature leads to the formation of cavities similar to those observed when the hydrogenation is performed in the presence of gaseous hydrogen at high pressure and temperature (CW)

  9. Sorption of cobalt and nickel on anaerobic granular sludges: isotherms and sequential extraction

    NARCIS (Netherlands)

    Hullebusch, van E.D.; Peerbolte, A.; Zandvoort, M.H.; Lens, P.N.L.

    2005-01-01

    The objective of this study was to investigate the sorption capacity and the fractionation of sorbed nickel and cobalt onto anaerobic granular sludges. Two different anaerobic granular sludges (non-fed, pH = 7) were loaded with nickel and cobalt in. adsorption experiments (monometal and competitive

  10. Tandem Affinity Purification Approach Coupled to Mass Spectrometry to Identify Post-translational Modifications of Histones Associated with Chromatin-Binding Proteins.

    Science.gov (United States)

    Beyer, Sophie; Robin, Philippe; Ait-Si-Ali, Slimane

    2017-01-01

    Protein purification by tandem affinity purification (TAP)-tag coupled to mass spectrometry analysis is usually used to reveal protein complex composition. Here we describe a TAP-tag purification of chromatin-bound proteins along with associated nucleosomes, which allow exhaustive identification of protein partners. Moreover, this method allows exhaustive identification of the post-translational modifications (PTMs) of the associated histones. Thus, in addition to partner characterization, this approach reveals the associated epigenetic landscape that can shed light on the function and properties of the studied chromatin-bound protein.

  11. Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine.

    Directory of Open Access Journals (Sweden)

    Utut Widyastuti Suharsono

    2008-11-01

    Full Text Available Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE.

  12. Inhibitory GTP binding protein G/sub i/ regulates β-adrenoceptor affinity towards β-agonists

    International Nuclear Information System (INIS)

    Marbach, I.; Levitzki, A.

    1987-01-01

    Treatment of S-49 lymphoma cell membranes with pertussis toxin (PT) causes a three-fold reduction of β-adrenoceptor (βAR) affinity towards isoproterenol. A similar treatment with cholera toxin (CT) does not cause such a modulation. The effects were studied by the detailed analysis of 125 I-cyanopindolol (CYP) binding curves in the absence and presence of increasing agonist concentrations. Thus, the authors were able to compare in detail the effects of G/sub s/ and G/sub i/ on the agonist-associated state of the βAR. In contrast to these findings, PT treatment does not have any effect on the displacement of 125 I-CYP by (-)isoproterenol. These results demonstrate that the inhibitory GTP protein G/sub i/ modulates the βAR affinity towards β-agonists. This might be due to the association of G/sub i/ with the agonist-bound βAR x G/sub s/ x C complex within the membrane. This hypothesis, as well as others, is under investigation

  13. Recent improvements to Binding MOAD: a resource for protein-ligand binding affinities and structures.

    Science.gov (United States)

    Ahmed, Aqeel; Smith, Richard D; Clark, Jordan J; Dunbar, James B; Carlson, Heather A

    2015-01-01

    For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein-ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23,269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Affinity reagent technology development and application to rapid immunochromatographic pathogen detection

    Science.gov (United States)

    Sooter, Letha J.; Stratis-Cullum, Dimitra N.; Zhang, Yanting; Daugherty, Patrick S.; Soh, H. Tom; Pellegrino, Paul; Stagliano, Nancy

    2007-09-01

    Immunochromatography is a rapid, reliable, and cost effective method of detecting biowarfare agents. The format is similar to that of an over-the-counter pregnancy test. A sample is applied to one end of a cassette and then a control line, and possibly a sample line, are visualized at the other end of the cassette. The test is based upon a sandwich assay. For the control, a line of Protein A is immobilized on the membrane. Gold nanoparticle bound IgG flows through the membrane and binds the Protein A, creating a visible line on the membrane. For the sample, one epitope is immobilized on the membrane and another epitope is attached to gold nanoparticles. The sample binds gold bound epitope, travels through the membrane, and binds membrane bound epitope. The two epitopes are not cross-reactive, therefore a sample line is only visible if the sample is present. In order to efficiently screen for binders to a sample target, a novel, Continuous Magnetic Activated Cell Sorter (CMACS) has been developed on a disposable, microfluidic platform. The CMACS chip quickly sorts E. coli peptide libraries for target binders with high affinity. Peptide libraries, are composed of approximately ten million bacteria, each displaying a different peptide on their surface. The target of interest is conjugated to a micrometer sized magnetic particle. After the library and the target are incubated together to allow binding, the mixture is applied to the CMACS chip. In the presence of patterned nickel and an external magnet, separation occurs of the bead-bound bacteria from the bulk material. The bead fraction is added to bacterial growth media where any attached E. coli grow and divide. These cells are cloned, sequenced, and the peptides are assayed for target binding affinity. As a proof-of-principle, assays were developed for human C-reactive protein. More defense relevant targets are currently being pursued.

  15. Pd-Pt loaded graphene aerogel on nickel foam composite as binder-free anode for a direct glucose fuel cell unit

    Science.gov (United States)

    Tsang, Chi Him A.; Leung, D. Y. C.

    2017-09-01

    Fabrication of electrocatalyst for direct glucose fuel cell (DGFC) operation involves destructive preparation methods with the use of stabilizer like binder, which may cause activity depreciation. Binder-free electrocatalytic electrode becomes a possible solution to the above problem. Binder-free bimetallic Pd-Pt loaded graphene aerogel on nickel foam plates with different Pd/Pt ratios (1:2.32, 1:1.62, and 1:0.98) are successfully fabricated through a green one-step mild reduction process producing a Pd-Pt/GO/nickel form plate (NFP) composite. Anode with the binder-free electrocatalysts exhibit a strong activity in a batch type DGFC unit under room temperature. The effects of glucose and KOH concentrations, and the Pd/Pt ratios of the electrocatalyst on the DGFC performance are also studied. Maximum power density output of 1.25 mW cm-2 is recorded with 0.5 M glucose/3 M KOH as the anodic fuel, and Pd1Pt0.98/GA/NFP as catalyst, which is the highest obtained so far among other types of electrocatalyst.

  16. Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    2018-02-01

    Full Text Available New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus. In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of KD = 20 ± 1 nM.

  17. Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A.

    Science.gov (United States)

    Stoltenburg, Regina; Strehlitz, Beate

    2018-02-24

    New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus . In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of K D = 20 ± 1 nM.

  18. Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

    Science.gov (United States)

    Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide

    2010-02-15

    The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

  19. Selective induction of high-ouabain-affinity isoform of Na sup + -K sup + -ATPase by thyroid hormone

    Energy Technology Data Exchange (ETDEWEB)

    Haber, R.S.; Loeb, J.N. (Columbia Univ., New York, NY (USA))

    1988-12-01

    The administration of thyroid hormone is known to result in an induction of the Na{sup +}-K{sup +}-adenosinetriphosphatase (Na{sup +}-K{sup +}-ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissues. Since the Na{sup +}-K{sup +}-ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain. The authors have studied the effects of 3,3{prime},5-triiodo-L-thyronine (T{sub 3}) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na{sup +}-K{sup +}-ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (K{sub I}s) for ouabain of {approximately}10{sup {minus}7} and 10{sup {minus}4} M, respectively. Measurement of the specific binding of ({sup 3}H)ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (K{sub d}) of slightly less than 10{sup {minus}7}M, whose maximal binding capacity was increased by T{sub 3} treatment by 185%. Binding studies in unfractionated homogenates of diaphragm similarly demonstrated the presence of high-affinity sites whose maximal binding capacity was increased by T{sub 3} treatment. Quantitation of both the high- and low-ouabain-affinity forms of the Na{sup +}-K{sup +}-ATPase by ouabain-dependent phosphorylation from ({sup 32}P)orthophosphate confirmed that T{sub 3} treatment markedly increased the number of high-affinity sites while having little effect on the number of low-affinity sites. These observations provide, to our knowledge, the first demonstration that these two forms of the Na{sup +}-K{sup +}-ATPase are subject to selective hormonal induction.

  20. GMDH-Based Semi-Supervised Feature Selection for Electricity Load Classification Forecasting

    Directory of Open Access Journals (Sweden)

    Lintao Yang

    2018-01-01

    Full Text Available With the development of smart power grids, communication network technology and sensor technology, there has been an exponential growth in complex electricity load data. Irregular electricity load fluctuations caused by the weather and holiday factors disrupt the daily operation of the power companies. To deal with these challenges, this paper investigates a day-ahead electricity peak load interval forecasting problem. It transforms the conventional continuous forecasting problem into a novel interval forecasting problem, and then further converts the interval forecasting problem into the classification forecasting problem. In addition, an indicator system influencing the electricity load is established from three dimensions, namely the load series, calendar data, and weather data. A semi-supervised feature selection algorithm is proposed to address an electricity load classification forecasting issue based on the group method of data handling (GMDH technology. The proposed algorithm consists of three main stages: (1 training the basic classifier; (2 selectively marking the most suitable samples from the unclassified label data, and adding them to an initial training set; and (3 training the classification models on the final training set and classifying the test samples. An empirical analysis of electricity load dataset from four Chinese cities is conducted. Results show that the proposed model can address the electricity load classification forecasting problem more efficiently and effectively than the FW-Semi FS (forward semi-supervised feature selection and GMDH-U (GMDH-based semi-supervised feature selection for customer classification models.

  1. A peptide affinity column for the identification of integrin alpha IIb-binding proteins.

    Science.gov (United States)

    Daxecker, Heide; Raab, Markus; Bernard, Elise; Devocelle, Marc; Treumann, Achim; Moran, Niamh

    2008-03-01

    To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

  2. Exploiting the high-affinity phosphonate-hydroxyapatite nanoparticle interaction for delivery of radiation and drugs

    International Nuclear Information System (INIS)

    Ong, Hooi Tin; Loo, Joachim S. C.; Boey, Freddy Y. C.; Russell, Stephen J.; Ma Jan; Peng, Kah-Whye

    2008-01-01

    Hydroxyapatite is biocompatible and used in various biomedical applications. Here, we generated hydroxyapatite nanoparticles (HNPs) of various sizes (40-200 nm) and demonstrated that they can be stably loaded with drugs or radioisotopes by exploiting the high-affinity HA-(poly)phosphonate interaction. Clinically available phosphonates, clodronate, and Tc-99m-methylene-diphosphonate (Tc-99m-MDP), were efficiently loaded onto HNPs within 15 min. Biodistribution of radiolabeled HNP-MDP-Tc99m in mice was monitored non-invasively using microSPECT-CT. Imaging and dosimetry studies indicated that the HNPs, regardless of size, were quickly taken up by Kupffer cells in the liver after systemic administration into mice. Clodronate loaded onto HNPs remained biologically active and were able to result in selective depletion of Kupffer cells. This method of drug or isotope loading on HA is fast and easy as it eliminates the need for additional surface modifications of the nanoparticles

  3. Long-life fatigue test results for two nickel-base structural alloys

    International Nuclear Information System (INIS)

    Mowbray, D.F.; Giaquinto, E.V.; Mehringer, F.J.

    1978-11-01

    The results are reported of fatigue tests on two nickel--base alloys, hot-cold-worked and stress-relieved nickel--chrome--iron Alloy 600 and mill-annealed nickel--chrome--moly--iron Alloy 625 in which S-N data were obtained in the life range of 10 6 to 10 10 cycles. The tests were conducted in air at 600 0 F, in the reversed membrane loading mode, at a frequency of approx. 1850 Hz. An electromagnetic, closed loop servo-controlled machine was built to perform the tests. A description of the machine is given

  4. in silico identification of cross affinity towards Cry1Ac pesticidal protein with receptor enzyme in Bos taurus and sequence, structure analysis of crystal proteins for stability.

    Science.gov (United States)

    Ebenezer, King Solomon; Nachimuthu, Ramesh; Thiagarajan, Prabha; Velu, Rajesh Kannan

    2013-01-01

    Any novel protein introduced into the GM crops need to be evaluated for cross affinity on living organisms. Many researchers are currently focusing on the impact of Bacillus thuringiensis cotton on soil and microbial diversity by field experiments. In spite of this, in silico approach might be helpful to elucidate the impact of cry genes. The crystal a protein which was produced by Bt at the time of sporulation has been used as a biological pesticide to target the insectivorous pests like Cry1Ac for Helicoverpa armigera and Cry2Ab for Spodoptera sp. and Heliothis sp. Here, we present the comprehensive in silico analysis of Cry1Ac and Cry2Ab proteins with available in silico tools, databases and docking servers. Molecular docking of Cry1Ac with procarboxypeptidase from Helicoverpa armigera and Cry1Ac with Leucine aminopeptidase from Bos taurus has showed the 125(th) amino acid position to be the preference site of Cry1Ac protein. The structures were compared with each other and it showed 5% of similarity. The cross affinity of this toxin that have confirmed the earlier reports of ill effects of Bt cotton consumed by cattle.

  5. High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

    Science.gov (United States)

    Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

    2015-01-01

    SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

  6. High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

    Science.gov (United States)

    Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

    2015-02-13

    SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

    Science.gov (United States)

    Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

    2012-06-01

    Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

  8. Investigation of hydrogen evolution activity for the nickel, nickel-molybdenum nickel-graphite composite and nickel-reduced graphene oxide composite coatings

    International Nuclear Information System (INIS)

    Jinlong, Lv; Tongxiang, Liang; Chen, Wang

    2016-01-01

    Graphical abstract: - Highlights: • Improved HER efficiency of Ni-Mo coatings was attributed to ‘cauliflower’ like microstructure. • RGO in nickel-RGO composite coating promoted refined grain and facilitated HER. • Synergistic effect between nickel and RGO facilitated HER due to large specific surface of RGO. - Abstract: The nickel, nickel-molybdenum alloy, nickel-graphite and nickel-reduced graphene oxide composite coatings were obtained by the electrodeposition technique from a nickel sulfate bath. Nanocrystalline molybdenum, graphite and reduced graphene oxide in nickel coatings promoted hydrogen evolution reaction in 0.5 M H_2SO_4 solution at room temperature. However, the nickel-reduced graphene oxide composite coating exhibited the highest electrocatalytic activity for the hydrogen evolution reaction in 0.5 M H_2SO_4 solution at room temperature. A large number of gaps between ‘cauliflower’ like grains could decrease effective area for hydrogen evolution reaction in slight amorphous nickel-molybdenum alloy. The synergistic effect between nickel and reduced graphene oxide promoted hydrogen evolution, moreover, refined grain in nickel-reduced graphene oxide composite coating and large specific surface of reduced graphene oxide also facilitated hydrogen evolution reaction.

  9. Investigation of hydrogen evolution activity for the nickel, nickel-molybdenum nickel-graphite composite and nickel-reduced graphene oxide composite coatings

    Energy Technology Data Exchange (ETDEWEB)

    Jinlong, Lv, E-mail: ljlbuaa@126.com [Beijing Key Laboratory of Fine Ceramics, Institute of Nuclear and New Energy Technology, Tsinghua University, Zhongguancun Street, Haidian District, Beijing 100084 (China); State Key Lab of New Ceramic and Fine Processing, Tsinghua University, Beijing 100084 (China); Tongxiang, Liang; Chen, Wang [Beijing Key Laboratory of Fine Ceramics, Institute of Nuclear and New Energy Technology, Tsinghua University, Zhongguancun Street, Haidian District, Beijing 100084 (China); State Key Lab of New Ceramic and Fine Processing, Tsinghua University, Beijing 100084 (China)

    2016-03-15

    Graphical abstract: - Highlights: • Improved HER efficiency of Ni-Mo coatings was attributed to ‘cauliflower’ like microstructure. • RGO in nickel-RGO composite coating promoted refined grain and facilitated HER. • Synergistic effect between nickel and RGO facilitated HER due to large specific surface of RGO. - Abstract: The nickel, nickel-molybdenum alloy, nickel-graphite and nickel-reduced graphene oxide composite coatings were obtained by the electrodeposition technique from a nickel sulfate bath. Nanocrystalline molybdenum, graphite and reduced graphene oxide in nickel coatings promoted hydrogen evolution reaction in 0.5 M H{sub 2}SO{sub 4} solution at room temperature. However, the nickel-reduced graphene oxide composite coating exhibited the highest electrocatalytic activity for the hydrogen evolution reaction in 0.5 M H{sub 2}SO{sub 4} solution at room temperature. A large number of gaps between ‘cauliflower’ like grains could decrease effective area for hydrogen evolution reaction in slight amorphous nickel-molybdenum alloy. The synergistic effect between nickel and reduced graphene oxide promoted hydrogen evolution, moreover, refined grain in nickel-reduced graphene oxide composite coating and large specific surface of reduced graphene oxide also facilitated hydrogen evolution reaction.

  10. Mycobacterium tuberculosis nucleoid-associated DNA-binding protein H-NS binds with high-affinity to the Holliday junction and inhibits strand exchange promoted by RecA protein.

    Science.gov (United States)

    Sharadamma, N; Harshavardhana, Y; Singh, Pawan; Muniyappa, K

    2010-06-01

    A number of studies have shown that the structure and composition of bacterial nucleoid influences many a processes related to DNA metabolism. The nucleoid-associated proteins modulate not only the DNA conformation but also regulate the DNA metabolic processes such as replication, recombination, repair and transcription. Understanding of how these processes occur in the context of Mycobacterium tuberculosis nucleoid is of considerable medical importance because the nucleoid structure may be constantly remodeled in response to environmental signals and/or growth conditions. Many studies have concluded that Escherichia coli H-NS binds to DNA in a sequence-independent manner, with a preference for A-/T-rich tracts in curved DNA; however, recent studies have identified the existence of medium- and low-affinity binding sites in the vicinity of the curved DNA. Here, we show that the M. tuberculosis H-NS protein binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double-stranded DNA with relatively higher GC content. Notably, M. tuberculosis H-NS was able to bind Holliday junction (HJ), the central recombination intermediate, with substantially higher affinity and inhibited the three-strand exchange promoted by its cognate RecA. Likewise, E. coli H-NS was able to bind the HJ and suppress DNA strand exchange promoted by E. coli RecA, although much less efficiently compared to M. tuberculosis H-NS. Our results provide new insights into a previously unrecognized function of H-NS protein, with implications for blocking the genome integration of horizontally transferred genes by homologous and/or homeologous recombination.

  11. Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

    Science.gov (United States)

    Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

    2007-08-01

    INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

  12. Comprehensive Characterization of Minichromosome Maintenance Complex (MCM) Protein Interactions Using Affinity and Proximity Purifications Coupled to Mass Spectrometry.

    Science.gov (United States)

    Dubois, Marie-Line; Bastin, Charlotte; Lévesque, Dominique; Boisvert, François-Michel

    2016-09-02

    The extensive identification of protein-protein interactions under different conditions is an important challenge to understand the cellular functions of proteins. Here we use and compare different approaches including affinity purification and purification by proximity coupled to mass spectrometry to identify protein complexes. We explore the complete interactome of the minichromosome maintenance (MCM) complex by using both approaches for all of the different MCM proteins. Overall, our analysis identified unique and shared interaction partners and proteins enriched for distinct biological processes including DNA replication, DNA repair, and cell cycle regulation. Furthermore, we mapped the changes in protein interactions of the MCM complex in response to DNA damage, identifying a new role for this complex in DNA repair. In summary, we demonstrate the complementarity of these approaches for the characterization of protein interactions within the MCM complex.

  13. Lifetime assessment of thick-walled components made of nickel-base alloys under near-service loading conditions

    International Nuclear Information System (INIS)

    Hueggenberg, Daniel

    2015-01-01

    Until 2050 the renewable energies should provide 80% of the power in Germany according to Renewable Energy law. Due to that reason the conventional power plants are not used for base load, but rather for the supply of average and peak load. The change of the operating mode leads to shorter times at stationary temperatures and the number of faster start-ups/shut-downs of the power plants will increase. As a result of this the components are exposed to an interacting load of creep and fatigue which reduces the lifetimes. The aim of this thesis is the development and verification of a lifetime assessment procedure for components made of the nickel-base alloys Alloy 617 mod. and Alloy 263 under creep fatigue loading conditions based on numerical phenomenological models and on the approaches of different standards/recommendations. The focus lies on two components of the high temperature material test rig II (HWT II), a header made of Alloy 617 mod. and Alloy 263 as well as a formed part made of Alloy 617 mod. For the basis characterization of the HWT II melts, specimens of the Alloy 617 mod. and Alloy 263 are tested in uniaxial tensile tests, (creep-)fatigue tests, creep tests and charpy tests in a temperature range between 20 C and 725 C. From the comparisons of the test results and the material specifications respectively the results of the projects COORETEC DE4, MARCKO DE2 and MARCKO700 no deviations were obvious for both materials with the exception of the creep test results with Alloy 617 mod. material. The creep tests with Alloy 617 mod. material of the HWT II melt show differences regarding the deformation and damage behavior. In addition to the basis characterization tests some complex lab tests for the characterization of the material behavior under creep-fatigue and multiaxial loading conditions were conducted. The developments of the microstructure, the precipitations as well as the structure of dislocations are investigated in the light optical microscope

  14. Mutual Information-Based Inputs Selection for Electric Load Time Series Forecasting

    Directory of Open Access Journals (Sweden)

    Nenad Floranović

    2013-02-01

    Full Text Available Providing accurate load forecast to electric utility corporations is essential in order to reduce their operational costs and increase profits. Hence, training set selection is an important preprocessing step which has to be considered in practice in order to increase the accuracy of load forecasts. The usage of mutual information (MI has been recently proposed in regression tasks, mostly for feature selection and for identifying the real instances from training sets that contains noise and outliers. This paper proposes a methodology for the training set selection in a least squares support vector machines (LS-SVMs load forecasting model. A new application of the concept of MI is presented for the selection of a training set based on MI computation between initial training set instances and testing set instances. Accordingly, several LS-SVMs models have been trained, based on the proposed methodology, for hourly prediction of electric load for one day ahead. The results obtained from a real-world data set indicate that the proposed method increases the accuracy of load forecasting as well as reduces the size of the initial training set needed for model training.

  15. Determination of nickel in hydrogenated fats and selected chocolate bars in Czech Republic.

    Science.gov (United States)

    Dohnalova, Lucie; Bucek, Pavel; Vobornik, Petr; Dohnal, Vlastimil

    2017-02-15

    Nickel is a metal that can be present in products containing hardened edible oils, possibly as leftover catalyst from the vegetable oil hardening process. Nickel may cause toxic effects including the promotion of cancer and contact allergy. In this work, nickel content was determined in hydrogenated vegetable fats and confectionery products, made with these fats, available on the Czech market using newly developed method combining microwave digestion and graphite furnace AAS. While concentrations of 0.086±0.014mg.kg(-1) or less were found in hydrogenated vegetable fats, the Ni content in confectionery products was significantly higher, varying between 0.742±0.066 and 3.141±0.217mg.kg(-1). Based on an average consumer basket, daily intake of nickel from vegetable fats is at least twice as low as intake from confectionery products. Based on results, the levels of nickel in neither vegetable fats nor confectionery products, do not represent a significant health risk. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Perceptual load manipulation reveals sensitivity of the face-selective N170 to attention.

    Science.gov (United States)

    Mohamed, Tarik N; Neumann, Markus F; Schweinberger, Stefan R

    2009-05-27

    It has been controversial whether the face-sensitive N170 is affected by selective attention. We manipulated attention sensu Lavie's perceptual load theory to short (200 ms) presentations of task-irrelevant unfamiliar faces or houses, while participants identified superimposed target letters 'X' versus 'N'. These targets were strings of either six identical (low load) or six different letters (high load). Under low load, we found a prominent face-selective N170 response. Under high load, however, we not only observed a dramatic reduction of the face N170 but also an unexpected enhancement of the house N170, such that face selectivity was almost completely lost. We conclude that the early stages of face processing indexed by the N170 strongly depend on selective attention.

  17. The removal of toxic metals from liquid effluents by ion exchange resins. Part V: Nickel(II/H+/Dowex C400

    Directory of Open Access Journals (Sweden)

    Francisco José Alguacil

    2017-11-01

    Full Text Available The cationic exchange resin Dowex C400 was used to remove nickel(II from aqueous solutions of different pH values and under various experimental conditions: stirring speed of the aqueous solution/resin system, temperature, resin dosage and aqueous ionic strength. The selectivity of the resin was investigated against the presence of various metals in the aqueous solution, and the removal of nickel(II from aqueous solutions was also compared with results obtained using multiwalled carbon nanotubes or functionalized (carboxylic groups multiwalled carbon nanotubes as adsorbents. According to batch experimental data, best fit of the results is obtained with the Freundlich model, whereas the ion exchange process is best explained by the pseudo-first order model. Experimental data fit well to the moving boundary controlled model. Elution of the nickel(II loaded onto Dowex C400 resin is fully possible using acidic solutions.

  18. A new perspective on the perceptual selectivity of attention under load.

    Science.gov (United States)

    Giesbrecht, Barry; Sy, Jocelyn; Bundesen, Claus; Kyllingsbaek, Søren

    2014-05-01

    The human attention system helps us cope with a complex environment by supporting the selective processing of information relevant to our current goals. Understanding the perceptual, cognitive, and neural mechanisms that mediate selective attention is a core issue in cognitive neuroscience. One prominent model of selective attention, known as load theory, offers an account of how task demands determine when information is selected and an account of the efficiency of the selection process. However, load theory has several critical weaknesses that suggest that it is time for a new perspective. Here we review the strengths and weaknesses of load theory and offer an alternative biologically plausible computational account that is based on the neural theory of visual attention. We argue that this new perspective provides a detailed computational account of how bottom-up and top-down information is integrated to provide efficient attentional selection and allocation of perceptual processing resources. © 2014 New York Academy of Sciences.

  19. Blocking peptides against HBV: PreS1 protein selected from a phage display library

    International Nuclear Information System (INIS)

    Wang, Wei; Liu, Yang; Zu, Xiangyang; Jin, Rui; Xiao, Gengfu

    2011-01-01

    Highlights: → Successfully selected specific PreS1-interacting peptides by using phage displayed library. → Alignment of the positive phage clones revealed a consensus PreS1 binding motif. → A highly enriched peptide named P7 had a strong binding ability for PreS1. → P7 could block PreS1 attachment. -- Abstract: The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection. A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX n HX m HP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.

  20. Blocking peptides against HBV: PreS1 protein selected from a phage display library

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wei; Liu, Yang; Zu, Xiangyang; Jin, Rui [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China)

    2011-09-09

    Highlights: {yields} Successfully selected specific PreS1-interacting peptides by using phage displayed library. {yields} Alignment of the positive phage clones revealed a consensus PreS1 binding motif. {yields} A highly enriched peptide named P7 had a strong binding ability for PreS1. {yields} P7 could block PreS1 attachment. -- Abstract: The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection. A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX{sub n}HX{sub m}HP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.

  1. Amplification of the Ect2 proto-oncogene and over-expression of Ect2 mRNA and protein in nickel compound and methylcholanthrene-transformed 10T1/2 mouse fibroblast cell lines

    International Nuclear Information System (INIS)

    Clemens, Farrah; Verma, Rini; Ramnath, Jamuna; Landolph, Joseph R.

    2005-01-01

    Occupational exposure of humans to mixtures of insoluble and soluble nickel (Ni) compounds correlates with increased incidences of lung, sinus, and pharyngeal tumors. Specific insoluble Ni compounds are carcinogenic to animals by inhalation and induce morphological and neoplastic transformation of cultured rodent cells. Our objectives were to (1) understand mechanisms of nickel ion-induced cell transformation, hence carcinogenesis and (2) develop biomarkers of nickel ion exposure and nickel ion-induced cell transformation. We isolated mRNAs from green nickel oxide (NiO), crystalline nickel monosulfide (NiS), and 3-methylcholanthrene (MCA) transformed C3H/10T1/2 Cl 8 cell lines, and determined by mRNA differential display that nine mRNA fragments were differentially expressed between Ni transformed and non-transformed 10T1/2 cell lines. Fragment R2-5 was expressed at higher steady-state levels in the transformed cell lines. R2-5 had 100% sequence identity to part of the coding region of Ect2, a mouse proto-oncogene encoding a GDP-GTP exchange factor. The 3.9-kb Ect2 transcript was expressed at 1.6- to 3.6-fold higher steady-state levels in four Ni transformed, and in two MCA-transformed, cell lines. Ect2 protein was expressed at 3.0- to 4.5-fold higher steady-state levels in Ni-transformed and in MCA-transformed cell lines. The Ect2 gene was amplified by 3.5- to 10-fold in Ni transformed, and by 2.5- to 3-fold in MCA transformed cell lines. Binding of nickel ions to enzymes of DNA synthesis likely caused amplification of the Ect2 gene. Ect2 gene amplification and over-expression of Ect2 mRNA and protein can cause microtubule disassembly and cytokinesis, contributing to induction and maintenance of morphological, anchorage-independent, and neoplastic transformation of these cell lines. Over-expression of Ect2 protein is a useful biomarker to detect exposure to nickel compounds and nickel ion-induced morphological and neoplastic cell transformation

  2. Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein.

    Science.gov (United States)

    Osipiuk, J; Georgopoulos, C; Zylicz, M

    1993-03-05

    It is known that the initiation of bacteriophage lambda replication requires the orderly assembly of the lambda O.lambda P.DnaB helicase protein preprimosomal complex at the ori lambda DNA site. The DnaK, DnaJ, and GrpE heat shock proteins act together to destabilize the lambda P.DnaB complex, thus freeing DnaB and allowing it to unwind lambda DNA near the ori lambda site. The first step of this disassembly reaction is the binding of DnaK to the lambda P protein. In this report, we examined the influence of the DnaJ and GrpE proteins on the stability of the lambda P.DnaK complex. We present evidence for the existence of the following protein-protein complexes: lambda P.DnaK, lambda P.DnaJ, DnaJ.DnaK, DnaK.GrpE, and lambda P.DnaK.GrpE. Our results suggest that the presence of GrpE alone destabilizes the lambda P.DnaK complex, whereas the presence of DnaJ alone stabilizes the lambda P.DnaK complex. Using immunoprecipitation, we show that in the presence of GrpE, DnaK exhibits a higher affinity for the lambda P.DnaJ complex than it does alone. Using cross-linking with glutaraldehyde, we show that oligomeric forms of DnaK exhibit a higher affinity for lambda P than monomeric DnaK. However, in the presence of GrpE, monomeric DnaK can efficiently bind lambda P protein. These findings help explain our previous results, namely that in the GrpE-dependent lambda DNA replication system, the DnaK protein requirement can be reduced up to 10-fold.

  3. Novel trends in affinity biosensors: current challenges and perspectives

    International Nuclear Information System (INIS)

    Arugula, Mary A; Simonian, Aleksandr

    2014-01-01

    Molecular biorecognition processes facilitate physical and biochemical interactions between molecules in all crucial metabolic pathways. Perhaps the target analyte and the biorecognition element interactions have the most impactful use in biosensing applications. Traditional analytical sensing systems offer excellent biorecognition elements with the ability to detect and determine the presence of analytes. High affinity antibodies and DNA play an important role in the development of affinity biosensors based on electrochemical, optical and mass sensitive approaches. Advancements in this area routinely employ labels, label free, nanoparticles, multifunctional matrices, carbon nanotubes and other methods to meet the requirements of its own application. However, despite increasing affinity ceilings for conventional biosensors, the field draws back in meeting specifically important demands, such as long-term stability, ultrasensitivity, rapid detection, extreme selectivity, strong biological base, calibration, in vivo measurements, regeneration, satisfactory performance and ease of production. Nevertheless, recent efforts through this line have produced novel high-tech nanosensing systems such as ‘aptamers’ and ‘phages’ which exhibit high-throughput sensing. Aptamers and phages are powerful tools that excel over antibodies in sensibility, stability, multi-detection, in vivo measurements and regeneration. Phages are superior in stability, screening for affinity-based target molecules ranging from small to proteins and even cells, and easy production. In this review, we focus mainly on recent developments in affinity-based biosensors such as immunosensors, DNA sensors, emphasizing aptasensors and phage-based biosensors basing on novel electrochemical, optical and mass sensitive detection techniques. We also address enzyme inhibition-based biosensors and the current problems associated with the above sensors and their future perspectives. (topical review)

  4. Slip activity of persistent slip bands in polycrystalline nickel

    International Nuclear Information System (INIS)

    Weidner, A.; Beyer, R.; Blochwitz, C.; Holste, C.; Schwab, A.; Tirschler, W.

    2006-01-01

    The appearance of glide localizations after cyclic deformation in the saturation stage was investigated for polycrystalline nickel. It was shown that persistent slip bands (PSBs) are formed in a wide range of grain orientations. Concerning the grain size it was found, that the probability for the appearance of PSBs is higher for larger grains. The local slip activity of the formed PSBs was studied after half-cycle deformation using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The fraction of grains with glide-active PSBs and the glide-active PSB volume itself is very small after the half-cycle loading. The obtained local shear strain amplitudes are quite high and vary in the range of 0.2-5%. They are comparable with those found in nickel single crystals at the same loading procedure

  5. Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography

    Directory of Open Access Journals (Sweden)

    Lina Lu

    2015-05-01

    Full Text Available Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed.

  6. Mercury and nickel contents in fish meat

    Directory of Open Access Journals (Sweden)

    Tomas Toth

    2016-10-01

    Full Text Available The main aim of our work was to identify the content of mercury and nickel in selected fish species. Consumers today are increasingly aware of the association between diet and health, and thus in Europe consume more and more fish. Fish is a valuable source of high quality protein, minerals and vitamins, and fatty fish are also rich in omega-3-polyunsaturated fatty acids, which are normally considered to be beneficial to health. In our work we determined content of mercury and nickel harvested fish in particular were the following species: Common goldfish (Carassius auratus, L., Common roach (Rutilus rutilus, L. and Common bream (Abramis brama, L. Concentrations of mercury and nickel was analyzed and results evaluated according to current standards and compared to the values ​​established by the Codex Alimentarius of the Slovak Republic and the EU Commission Regulation no. 1881/2006, as well as in the EU Commission Regulation no. 420/2011 and no. 269/2008. In our research area we analysed 19 samples of fish muscle. Samples were taken from two water reservoirs – Golianovo and Vráble.  The highest mercury content was in sample Rutilus 1 - 0.052632 mg/kg. Lowest mercury content was in sample Abramis 2  - 0.010431 mg/kg. Largest nickel content was in meat of Abramis  - sample 2  - 0.78 mg/kg. Minimum content of nickel was in sample Carassius 1  - 0.11 mg/kg. We got out of the limit values ​​specified: Codex Alimentarius SR - Mercury 0.5 mg/kg and  Regulation of the EU Commission no. 1881/2006, no. 420/2011 and no. 629/2008. To optimize the protection of the population, it is necessary to continue to monitor the concentration of mercury in fish and fish products. Risk management strategy must focus on reducing potential exposure derived from consumption of fish. In particular, the definition of maximum levels for methylmercury, advising consumers and environmental activities oriented to reduce contamination.

  7. Water-in-Oil Microemulsions for Protein Delivery: Loading Optimization and Stability.

    Science.gov (United States)

    Perinelli, Diego R; Cespi, Marco; Pucciarelli, Stefania; Vincenzetti, Silvia; Casettari, Luca; Lam, Jenny K W; Logrippo, Serena; Canala, Elisa; Soliman, Mahmoud E; Bonacucina, Giulia; Palmieri, Giovanni F

    2017-01-01

    Microemulsions are attractive delivery systems for therapeutic proteins and peptides due to their ability to enhance bioavailability. Although different proteins and peptides have been successfully delivered through such ternary systems, no information can be found about protein loading and the formulation stability when such microemulsions are prepared with pharmaceuticallyapproved oils and surfactants. The aim of this work was to optimise a ternary system consisting of water/ ethyl oleate/Span® 80-Tween® 80 and to determine its protein loading capacity and stability, using bovine serum albumin (BSA) as a model of biomolecule. The optimization was carried out using a Central Composite Design and all the prepared formulations were characterised through dynamic light scattering, rheology, optical and polarized microscopy. Subsequently, the maximum loading capacity was determined and the stability of the final microemulsion with the highest content of protein was followed over six months. To investigate the structural features of the protein, BSA was recovered from the microemulsion and analysed through fluorescence spectroscopy. After incorporation of the protein in the microemulsion, a decrease of its aqueous solubility was observed. However, the formulation remained stable over six months and the native-like state of the recovered protein was demonstrated by fluorescence spectroscopy Conclusion: This study demonstrated the feasibility of preparing microemulsions with the highest content of protein and their long-term stability. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  8. Preparation and characterization of an organic/inorganic hybrid sorbent (PLE) to enhance selectivity for As(V).

    Science.gov (United States)

    An, Byungryul; Kim, Hakchan; Park, Chanhyuk; Lee, Sang-Hyup; Choi, Jae-Woo

    2015-05-30

    For the selective removal of arsenate (As(V)) a hybrid sorbent was prepared using a non-toxic natural organic material, chitosan, by loading a transition metal, nickel. The immobilization of nickel was achieved by coordination with a deprotonated amino group (NH2) in the chitosan polymer chain. The amount of nickel was directly correlated to the presence of the amino group and was calculated to be 62 mg/g. FTIR spectra showed a peak shift from 1656 to 1637 cm(-1) after Ni(2+) loading, indicating the complexation between the amino group and nickel, and a peak of As(V) was observed at 834 cm(-1). An increase of sulfate concentration from 100 mg/L to 200 mg/L did not significantly affect As(V) sorption, and an increase in the concentration of bicarbonate reduced the As(V) uptake by 33%. The optimal pH of the solution was determined at pH 10, which is in accordance with the fraction of HAsO4(2-) and AsO4(-3). According to a fixed column test, a break through behavior of As(V) revealed that selectivity for As(V) was over sulfate. Regeneration using 5% NaCl extended the use of sorbent to up to uses without big loss of sorption capacity. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. A new perspective on the perceptual selectivity of attention under load

    DEFF Research Database (Denmark)

    Giesbrecht, Barry; Sy, Jocelyn; Bundesen, Claus

    2014-01-01

    The human attention system helps us cope with a complex environment by supporting the selective processing of information relevant to our current goals. Understanding the perceptual, cognitive, and neural mechanisms that mediate selective attention is a core issue in cognitive neuroscience. One...... prominent model of selective attention, known as load theory, offers an account of how task demands determine when information is selected and an account of the efficiency of the selection process. However, load theory has several critical weaknesses that suggest that it is time for a new perspective. Here...... is integrated to provide efficient attentional selection and allocation of perceptual processing resources....

  10. Capillary electrophoresis-based assessment of nanobody affinity and purity

    NARCIS (Netherlands)

    Haselberg, Rob; Oliveira, Sabrina; van der Meel, Roy; Somsen, Govert W; de Jong, Gerhardus J

    2014-01-01

    Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced "nanobody" EGa1, the binding fragment of a

  11. Using affinity capillary electrophoresis and computational models for binding studies of heparinoids with p-selectin and other proteins.

    Science.gov (United States)

    Mozafari, Mona; Balasupramaniam, Shantheya; Preu, Lutz; El Deeb, Sami; Reiter, Christian G; Wätzig, Hermann

    2017-06-01

    A fast and precise affinity capillary electrophoresis (ACE) method has been developed and applied for the investigation of the binding interactions between P-selectin and heparinoids as potential P-selectin inhibitors in the presence and absence of calcium ions. Furthermore, model proteins and vitronectin were used to appraise the binding behavior of P-selectin. The normalized mobility ratios (∆R/R f ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. It was found that P-selectin interacts more strongly with heparinoids in the presence of calcium ions. P-selectin was affected by heparinoids at the concentration of 3 mg/L. In addition, the results of the ACE experiments showed that among other investigated proteins, albumins and vitronectin exhibited strong interactions with heparinoids. Especially with P-selectin and vitronectin, the interaction may additionally induce conformational changes. Subsequently, computational models were applied to interpret the ACE experiments. Docking experiments explained that the binding of heparinoids on P-selectin is promoted by calcium ions. These docking models proved to be particularly well suited to investigate the interaction of charged compounds, and are therefore complementary to ACE experiments. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. High affinity binding of [3H]cocaine to rat liver microsomes

    International Nuclear Information System (INIS)

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    ] 3 H]cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in [ 3 H]cocaine binding. On the other hand, chronic administration of cocaine reduced [ 3 H]cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of [ 3 H]cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced [ 3 H]cocaine binding to liver with a different rank order of potency than their displacement of [ 3 H]cocaine binding to striatum. This high affinity [ 3 H]cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  13. Stapled Voltage-Gated Calcium Channel (CaV) α-Interaction Domain (AID) Peptides Act As Selective Protein-Protein Interaction Inhibitors of CaV Function.

    Science.gov (United States)

    Findeisen, Felix; Campiglio, Marta; Jo, Hyunil; Abderemane-Ali, Fayal; Rumpf, Christine H; Pope, Lianne; Rossen, Nathan D; Flucher, Bernhard E; DeGrado, William F; Minor, Daniel L

    2017-06-21

    For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein-protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein-protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein-protein interaction, the interaction between the voltage-gated calcium channel (Ca V ) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (Ca V β). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:Ca V β interactions and reduce the entropic penalty associated with AID binding to Ca V β. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the Ca V α 1 :Ca V β interaction that modulate Ca V function in an Ca V β isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein-protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based Ca V modulator design.

  14. Absolute determination by X-ray diffraction of a binary or ternary mixture: nickel oxide and fluoride in a nickel powder (1960); Dosage absolu par diffraction X d'un melange binaire ou ternaire: oxyde et fluorure de nickel dans une poudre de nickel (1960)

    Energy Technology Data Exchange (ETDEWEB)

    Charpin, P; Hauptman, A [Commissariat a l' Energie Atomique, Saclay (France).Centre d' Etudes Nucleaires

    1960-07-01

    The method employed is based upon the comparison between computed and measured intensities for conveniently selected X-Ray diffraction lines of each component of the powder. Care must be taken to allow for absorption, both inside each grain and in overall sample. This method has been applied to the determination of nickel oxide and fluoride in a nickel powder. (author) [French] La methode utilisee, dite 'absolue' est basee sur le calcul des intensites theoriques de raies de diffraction convenablement choisies. Elle n'est applicable que si l'absorption est negligeable a travers chaque grain constituant l'echantillon et a travers l'echantillon total. Elle a ete employee pour doser, ensemble ou separement, de l'oxyde et du fluorure de nickel dans une poudre de nickel. (auteur)

  15. Microfluidics in the selection of affinity reagents for the detection of cancer: paving a way towards future diagnostics.

    Science.gov (United States)

    Hung, Lien-Yu; Wang, Chih-Hung; Fu, Chien-Yu; Gopinathan, Priya; Lee, Gwo-Bin

    2016-08-07

    Microfluidic technologies have miniaturized a variety of biomedical applications, and these chip-based systems have several significant advantages over their large-scale counterparts. Recently, this technology has been used for automating labor-intensive and time-consuming screening processes, whereby affinity reagents, including aptamers, peptides, antibodies, polysaccharides, glycoproteins, and a variety of small molecules, are used to probe for molecular biomarkers. When compared to conventional methods, the microfluidic approaches are faster, more compact, require considerably smaller quantities of samples and reagents, and can be automated. Furthermore, they allow for more precise control of reaction conditions (e.g., pH, temperature, and shearing forces) such that more efficient screening can be performed. A variety of affinity reagents for targeting cancer cells or cancer biomarkers are now available and will likely replace conventional antibodies. In this review article, the selection of affinity reagents for cancer cells or cancer biomarkers on microfluidic platforms is reviewed with the aim of highlighting the utility of such approaches in cancer diagnostics.

  16. Identification of Thioredoxin Target Disulfides Using Isotope-Coded Affinity Tags

    DEFF Research Database (Denmark)

    Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

    2014-01-01

    Thioredoxins (Trx) are small redox proteins that reduce disulfide bonds in various target proteins and maintain cellular thiol redox control. Here, a thiol-specific labeling and affinity enrichment approach for identification and relative quantification of Trx target disulfides in complex protein...... reduction is determined by LC-MS/MS-based quantification of tryptic peptides labeled with "light" (12C) and "heavy" (13C) ICAT reagents. The methodology can be adapted to monitor the effect of different reductants or oxidants on the redox status of thiol/disulfide proteomes in biological systems....... extracts is described. The procedure utilizes the isotope-coded affinity tag (ICAT) reagents containing a thiol reactive iodoacetamide group and a biotin affinity tag to target peptides containing reduced cysteine residues. The identification of substrates for Trx and the extent of target disulfide...

  17. Fanconi anemia complementation group A (FANCA) protein has intrinsic affinity for nucleic acids with preference for single-stranded forms.

    Science.gov (United States)

    Yuan, Fenghua; Qian, Liangyue; Zhao, Xinliang; Liu, Jesse Y; Song, Limin; D'Urso, Gennaro; Jain, Chaitanya; Zhang, Yanbin

    2012-02-10

    The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5'-flap or 5'-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772-1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found.

  18. Fanconi Anemia Complementation Group A (FANCA) Protein Has Intrinsic Affinity for Nucleic Acids with Preference for Single-stranded Forms*

    Science.gov (United States)

    Yuan, Fenghua; Qian, Liangyue; Zhao, Xinliang; Liu, Jesse Y.; Song, Limin; D'Urso, Gennaro; Jain, Chaitanya; Zhang, Yanbin

    2012-01-01

    The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5′-flap or 5′-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772–1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found. PMID:22194614

  19. Lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (LIMACS): a novel method to analyze protein-lipid interaction.

    Science.gov (United States)

    Bieberich, Erhard

    2011-04-26

    The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids. To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane

  20. Reconstitution of high-affinity opioid agonist binding in brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Remmers, A.E.; Medzihradsky, F. (Univ. of Michigan Medical School, Ann Arbor (United States))

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  1. Manual for calculating critical loads of heavy metals for soils and surface waters; preliminary guidelines for environmental quality criteria, calculation methods and input data

    NARCIS (Netherlands)

    Vries, de W.; Bakker, D.J.

    1996-01-01

    Methodologies are described for calculating critical loads of lead, cadmium, copper, zinc, nickel, chromium and mercury for soils and surface waters. The aspects which are discussed are: selection of a computation model, determination of environmental-quality criteria for the metals, collection of

  2. Characterization of closed nickel-titanium orthodontic coil springs

    Energy Technology Data Exchange (ETDEWEB)

    Langeron, T. [Rene Descartes Univ., Paris V, Pontoise (France). Faculte de Chirurgie Dentaire; Filleul, M.P. [Rene Descartes Univ., Paris V, Pontoise (France). Faculte de Chirurgie Dentaire; ENSCP, Paris (France). Lab. de Metallurgie Structurale; Humbeeck, J. van [Katholieke Univ. Leuven, Heverlee (Belgium). Faculteit Toegepaste Wetenschappen, Metaalkunde en Toegepaste Materialkund

    2001-11-01

    Nickel-titanium orthodontic coil springs are used to move teeth with low forces and slow deactivation. The present paper provides data on transformation temperatures and on load-deflection rate at buccal temperature of closed Nickel-Titanium coil springs available on the market from ORMCO {sup trademark} and GAC {sup trademark}. All the springs exhibited superelasticity but their properties were not stable in the range of buccal temperatures and varied not only from one manufacturer to the other but they also varied from one batch to the other of each supplier. The need for more stability is stressed. (orig.)

  3. Cognitive load during route selection increases reliance on spatial heuristics.

    Science.gov (United States)

    Brunyé, Tad T; Martis, Shaina B; Taylor, Holly A

    2018-05-01

    Planning routes from maps involves perceiving the symbolic environment, identifying alternate routes and applying explicit strategies and implicit heuristics to select an option. Two implicit heuristics have received considerable attention, the southern route preference and initial segment strategy. This study tested a prediction from decision-making theory that increasing cognitive load during route planning will increase reliance on these heuristics. In two experiments, participants planned routes while under conditions of minimal (0-back) or high (2-back) working memory load. In Experiment 1, we examined how memory load impacts the southern route heuristic. In Experiment 2, we examined how memory load impacts the initial segment heuristic. Results replicated earlier results demonstrating a southern route preference (Experiment 1) and initial segment strategy (Experiment 2) and further demonstrated that evidence for heuristic reliance is more likely under conditions of concurrent working memory load. Furthermore, the extent to which participants maintained efficient route selection latencies in the 2-back condition predicted the magnitude of this effect. Together, results demonstrate that working memory load increases the application of heuristics during spatial decision making, particularly when participants attempt to maintain quick decisions while managing concurrent task demands.

  4. Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

    Energy Technology Data Exchange (ETDEWEB)

    Peabody, David S.; Chackerian, Bryce; Ashley, Carlee; Carnes, Eric; Negrete, Oscar

    2017-01-24

    The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referred to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.

  5. Evidence that the low-affinity folate-binding protein in erythrocyte hemolysate is identical to hemoglobin

    International Nuclear Information System (INIS)

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1981-01-01

    Gel filtration studies on erythrocyte hemolysate demonstrated the presence of a folate binding protein, apparently of the low-affinity type, that co-elutes with hemoglobin. Further, the folate binder eluted with a low salt concentration after DEAE-Sepharose CL-6B anion-exchange chromatography of erythrocyte hemolysate at pH 6.3. The chromatographic behavior of hemoglobin labeled with [3H]folate was so similar to that of the present binder as to suggest that the folate binder in erythrocytes is in fact hemoglobin

  6. Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin

    Directory of Open Access Journals (Sweden)

    Claire M. Gabe

    2017-06-01

    Full Text Available Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We

  7. Second Generation Grp94-Selective Inhibitors Provide Opportunities for the Inhibition of Metastatic Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Crowley, Vincent M. [Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Dr. Malott 4070 Lawrence KS 66045 USA; Huard, Dustin J. E. [School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta GA 30332 USA; Lieberman, Raquel L. [School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta GA 30332 USA; Blagg, Brian S. J. [Warren Family Research Center for Drug Discovery and Development, and Department of Chemistry & Biochemistry, University of Notre Dame, 305 McCourtney Hall Notre Dame IN 46556 USA

    2017-09-27

    Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum (ER) resident isoform of the 90 kDa heat shock protein (Hsp90) family and its inhibition represents a promising therapeutic target for the treatment of many diseases. Modification of the first generation cis-amide bioisostere imidazole to alter the angle between the resorcinol ring and the benzyl side chain via cis-amide replacements produced compounds with improved Grp94 affinity and selectivity. Structure–activity relationship studies led to the discovery of compound 30, which exhibits 540 nm affinity and 73-fold selectivity towards Grp94. Grp94 is responsible for the maturation and trafficking of proteins associated with cell signaling and motility, including select integrins. The Grp94-selective inhibitor 30 was shown to exhibit potent anti-migratory effects against multiple aggressive and metastatic cancers.

  8. Low affinity uniporter carrier proteins can increase net substrate uptake rate by reducing efflux

    NARCIS (Netherlands)

    Bosdriesz, Evert; Wortel, Meike T.; Haanstra, Jurgen R.; Wagner, Marijke J.; De La Torre Cortés, Pilar; Teusink, Bas

    2018-01-01

    Many organisms have several similar transporters with different affinities for the same substrate. Typically, high-affinity transporters are expressed when substrate is scarce and low-affinity ones when it is abundant. The benefit of using low instead of high-affinity transporters remains unclear,

  9. Low affinity uniporter carrier proteins can increase net substrate uptake rate by reducing efflux

    NARCIS (Netherlands)

    Bosdriesz, Evert; Wortel, M.T.; Haanstra, Jurgen R.; Wagner, Marijke J.; De La Torre, P.; Teusink, Bas

    2018-01-01

    Many organisms have several similar transporters with different affinities for the same substrate. Typically, high-affinity transporters are expressed when substrate is scarce and low-affinity ones when it is abundant. The benefit of using low instead of high-affinity transporters remains

  10. Experimental and theoretical binding affinity between polyvinylpolypyrrolidone and selected phenolic compounds from food matrices.

    Science.gov (United States)

    Durán-Lara, Esteban F; López-Cortés, Xaviera A; Castro, Ricardo I; Avila-Salas, Fabián; González-Nilo, Fernando D; Laurie, V Felipe; Santos, Leonardo S

    2015-02-01

    Polyvinylpolypyrrolidone (PVPP) is a fining agent, widely used in winemaking and brewing, whose mode of action in removing phenolic compounds has not been fully characterised. The aim of this study was to evaluate the experimental and theoretical binding affinity of PVPP towards six phenolic compounds representing different types of phenolic species. The interaction between PVPP and phenolics was evaluated in model solutions, where hydroxyl groups, hydrophobic bonding and steric hindrance were characterised. The results of the study indicated that PVPP exhibits high affinity for quercetin and catechin, moderate affinity for epicatechin, gallic acid and lower affinity for 4-methylcatechol and caffeic acid. The affinity has a direct correlation with the hydroxylation degree of each compound. The results show that the affinity of PVPP towards phenols is related with frontier orbitals. This work demonstrates a direct correlation between the experimental affinity and the interaction energy calculations obtained through computational chemistry methods. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Student award for outstanding research winner in the Ph.D. category for the 2017 society for biomaterials annual meeting and exposition, april 5-8, 2017, Minneapolis, Minnesota: Characterization of protein interactions with molecularly imprinted hydrogels that possess engineered affinity for high isoelectric point biomarkers.

    Science.gov (United States)

    Clegg, John R; Zhong, Justin X; Irani, Afshan S; Gu, Joann; Spencer, David S; Peppas, Nicholas A

    2017-06-01

    Molecularly imprinted polymers (MIPs) with selective affinity for protein biomarkers could find extensive utility as environmentally robust, cost-efficient biomaterials for diagnostic and therapeutic applications. In order to develop recognitive, synthetic biomaterials for prohibitively expensive protein biomarkers, we have developed a molecular imprinting technique that utilizes structurally similar, analogue proteins. Hydrogel microparticles synthesized by molecular imprinting with trypsin, lysozyme, and cytochrome c possessed an increased affinity for alternate high isoelectric point biomarkers both in isolation and plasma-mimicking adsorption conditions. Imprinted and non-imprinted P(MAA-co-AAm-co-DEAEMA) microgels containing PMAO-PEGMA functionalized polycaprolactone nanoparticles were net-anionic, polydisperse, and irregularly shaped. MIPs and control non-imprinted polymers (NIPs) exhibited regions of Freundlich and BET isotherm adsorption behavior in a range of non-competitive protein solutions, where MIPs exhibited enhanced adsorption capacity in the Freundlich isotherm regions. In a competitive condition, imprinting with analogue templates (trypsin, lysozyme) increased the adsorption capacity of microgels for cytochrome c by 162% and 219%, respectively, as compared to a 122% increase provided by traditional bulk imprinting with cytochrome c. Our results suggest that molecular imprinting with analogue protein templates is a viable synthetic strategy for enhancing hydrogel-biomarker affinity and promoting specific protein adsorption behavior in biological fluids. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1565-1574, 2017. © 2017 Wiley Periodicals, Inc.

  12. Development of an aptamer-based affinity purification method for vascular endothelial growth factor

    Directory of Open Access Journals (Sweden)

    Maren Lönne

    2015-12-01

    Full Text Available Since aptamers bind their targets with high affinity and specificity, they are promising alternative ligands in protein affinity purification. As aptamers are chemically synthesized oligonucleotides, they can be easily produced in large quantities regarding GMP conditions allowing their application in protein production for therapeutic purposes. Several advantages of aptamers compared to antibodies are described in general within this paper. Here, an aptamer directed against the human Vascular Endothelial Growth Factor (VEGF was used as affinity ligand for establishing a purification platform for VEGF in small scale. The aptamer was covalently immobilized on magnetic beads in a controlled orientation resulting in a functional active affinity matrix. Target binding was optimized by introduction of spacer molecules and variation of aptamer density. Further, salt-induced target elution was demonstrated as well as VEGF purification from a complex protein mixture proving the specificity of protein-aptamer binding.

  13. Selective Hydrogen Atom Abstraction through Induced Bond Polarization: Direct α-Arylation of Alcohols through Photoredox, HAT, and Nickel Catalysis.

    Science.gov (United States)

    Twilton, Jack; Christensen, Melodie; DiRocco, Daniel A; Ruck, Rebecca T; Davies, Ian W; MacMillan, David W C

    2018-05-04

    The combination of nickel metallaphotoredox catalysis, hydrogen atom transfer catalysis, and a Lewis acid activation mode, has led to the development of an arylation method for the selective functionalization of alcohol α-hydroxy C-H bonds. This approach employs zinc-mediated alcohol deprotonation to activate α-hydroxy C-H bonds while simultaneously suppressing C-O bond formation by inhibiting the formation of nickel alkoxide species. The use of Zn-based Lewis acids also deactivates other hydridic bonds such as α-amino and α-oxy C-H bonds. This approach facilitates rapid access to benzylic alcohols, an important motif in drug discovery. A 3-step synthesis of the drug Prozac exemplifies the utility of this new method. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Adaptive Equalizer Using Selective Partial Update Algorithm and Selective Regressor Affine Projection Algorithm over Shallow Water Acoustic Channels

    Directory of Open Access Journals (Sweden)

    Masoumeh Soflaei

    2014-01-01

    Full Text Available One of the most important problems of reliable communications in shallow water channels is intersymbol interference (ISI which is due to scattering from surface and reflecting from bottom. Using adaptive equalizers in receiver is one of the best suggested ways for overcoming this problem. In this paper, we apply the family of selective regressor affine projection algorithms (SR-APA and the family of selective partial update APA (SPU-APA which have low computational complexity that is one of the important factors that influences adaptive equalizer performance. We apply experimental data from Strait of Hormuz for examining the efficiency of the proposed methods over shallow water channel. We observe that the values of the steady-state mean square error (MSE of SR-APA and SPU-APA decrease by 5.8 (dB and 5.5 (dB, respectively, in comparison with least mean square (LMS algorithm. Also the families of SPU-APA and SR-APA have better convergence speed than LMS type algorithm.

  15. The endosomal sorting complex required for transport (ESCRT) is required for the sensitivity of yeast cells to nickel ions in Saccharomyces cerevisiae.

    Science.gov (United States)

    Luo, Chong; Cao, Chunlei; Jiang, Linghuo

    2016-05-01

    Nickel is one of the toxic environment metal pollutants and is linked to various human diseases. In this study, through a functional genomics approach we have identified 16 nickel-sensitive and 22 nickel-tolerant diploid deletion mutants of budding yeast genes, many of which are novel players in the regulation of nickel homeostasis. The 16 nickel-sensitive mutants are of genes mainly involved in the protein folding, modification and destination and the cellular transport processes, while the 22 nickel-tolerant mutants are of genes encoding components of ESCRT complexes as well as protein factors involved in both the cell wall integrity maintenance and the vacuolar protein sorting process. In consistence with their phenotypes, most of these nickel-sensitive mutants show reduced intracellular nickel contents, while the majority of these nickel-tolerant mutants show elevated intracellular nickel contents, as compared to the wild type in response to nickel stress. Our data provides a basis for our understanding the regulation of nickel homeostasis and molecular mechanisms of nickel-induced human pathogenesis. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Nickel toxicity on seed germination and growth in radish (Raphanus sativus) and its recovery using copper and boron.

    Science.gov (United States)

    Yadav, Shiv Shankar; Shukla, Rajni; Sharma, Y K

    2009-05-01

    Effect of various concentrations of nickel (100, 200, 500 and 1000 microM) and recovery treatments of boron (50 and 100 microM) and copper (15 and 75 microM) each with 200 microM and 500 microM of nickel on germination, growth, biomass, chlorophyll, carotenoids, pheophytin, amylase, protein, sugar as well as activity of catalase and peroxidase were studied in radish (Raphanus sativus cv. Early menu) seedlings. Nickel treatments caused a considerable reduction in germination percentage, growth and biomass. The different pigments were also decreased with nickel treatments. However boron addition with nickel recovered the negative effect on pigment contents. Among biochemical estimations, amylase activity and total proteins were found to be reduced in nickel treatments. Peroxidase and catalase activity were induced other than higher total sugar with nickel treatments. The combination of nickel with boron resulted into increased protein contents. This combination also reduced the catalase and peroxidase activity. The influence of nickel with copper failed to produce significant recovery except 200 microM nickel in combination with 15 microM copper with regard to catalase and peroxidase activity. The effect of nickel on hydrolyzing enzyme amylase was observed to be inhibitory resulting into poor germination followed by poor seedlings growth. The stress protecting enzymes peroxidase and catalase seem to be induced under the influence of nickel, and providing protection to the seedlings. The application of boron with nickel showed improved germination and growth. The level of catalase and peroxidase were found to be significantly reduced showing normal growth and biomass of seedlings.

  17. Nickel in nails, hair and plasma from nickel-hypersensitive women

    DEFF Research Database (Denmark)

    Gammelgaard, Bente; Veien, Niels

    1990-01-01

    The concentrations of nickel in finger-nails, toe-nails, hair and plasma from 71 nickel-hypersensitive women and 20 non-hypersensitive women were determined. Nickel concentrations in finger-nails were significantly higher than in toe-nails in both the nickel-hypersensitive group and the control...... group. Nickel-sensitive women had significantly higher levels of nickel in toe-nails, hair and plasma than had control subjects, whereas there was no significant difference in nickel concentration in finger-nails between the two groups. No correlation could be demonstrated between nickel levels in any...... combination of nails, hair and plasma in the nickel-hypersensitive or in the control group....

  18. Hydrogenation of Levulinic Acid over Nickel Catalysts Supported on Aluminum Oxide to Prepare γ-Valerolactone

    Directory of Open Access Journals (Sweden)

    Jie Fu

    2015-12-01

    Full Text Available Four types of nickel catalysts supported on aluminum oxide (Ni/Al2O3 with different nickel loadings were synthesized using the co-precipitation method and were used for the hydrogenation of levulinic acid (LA to prepare γ-valerolactone (GVL. The synthesized Ni/Al2O3 catalysts exhibited excellent catalytic activity in dioxane, and the activity of the catalysts was excellent even after being used four times in dioxane. The catalytic activity in dioxane as a solvent was found to be superior to the activity in water. Nitrogen physisorption, X-ray diffraction, and transmission electron microscopy were employed to characterize the fresh and used catalysts. The effects of the nickel loading, temperature, hydrogen pressure, and substrate/catalyst ratio on the catalytic activity were investigated.

  19. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    Science.gov (United States)

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-05-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method.

  20. Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue*

    Science.gov (United States)

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-01-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms—including humans—are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)1 with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

  1. Lattice-enabled nuclear reactions in the nickel and hydrogen gas system

    International Nuclear Information System (INIS)

    Nagel, David J.

    2015-01-01

    Thousands of lattice-enabled nuclear reaction (LENR) experiments involving electrochemical loading of deuterium into palladium have been conducted and reported in hundreds of papers. But, it appears that the first commercial LENR power generators will employ gas loading of hydrogen onto nickel. This article reviews the scientific base for LENR in the gas-loaded Ni-H system, and some of the tests of pre-commercial prototype generators based on this combination. (author)

  2. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    Science.gov (United States)

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

  3. Organic Microporous Nanofillers with Unique Alcohol Affinity for Superior Ethanol Recovery toward Sustainable Biofuels.

    Science.gov (United States)

    Cheng, Xi Quan; Konstas, Kristina; Doherty, Cara M; Wood, Colin D; Mulet, Xavier; Xie, Zongli; Ng, Derrick; Hill, Matthew R; Lau, Cher Hon; Shao, Lu

    2017-05-09

    To minimize energy consumption and carbon footprints, pervaporation membranes are fast becoming the preferred technology for alcohol recovery. However, this approach is confined to small-scale operations, as the flux of standard rubbery polymer membranes remain insufficient to process large solvent volumes, whereas membrane separations that use glassy polymer membranes are prone to physical aging. This study concerns how the alcohol affinity and intrinsic porosity of networked, organic, microporous polymers can simultaneously reduce physical aging and drastically enhance both flux and selectivity of a super glassy polymer, poly-[1-(trimethylsilyl)propyne] (PTMSP). Slight loss in alcohol transportation channels in PTMSP is compensated by the alcohol affinity of the microporous polymers. Even after continuous exposure to aqueous solutions of alcohols, PTMSP pervaporation membranes loaded with the microporous polymers outperform the state-of-the-art and commercial pervaporation membranes. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. The 100 kW space station. [regenerative fuel cells and nickel hydrogen and nickel cadmium batteries for solar arrays

    Science.gov (United States)

    Mckhann, G.

    1977-01-01

    Solar array power systems for the space construction base are discussed. Nickel cadmium and nickel hydrogen batteries are equally attractive relative to regenerative fuel cell systems at 5 years life. Further evaluation of energy storage system life (low orbit conditions) is required. Shuttle and solid polymer electrolyte fuel cell technology appears adequate; large units (approximately four times shuttle) are most appropriate and should be studied for a 100 KWe SCB system. A conservative NiH2 battery DOD (18.6%) was elected due to lack of test data and offers considerable improvement potential. Multiorbit load averaging and reserve capacity requirements limit nominal DOD to 30% to 50% maximum, independent of life considerations.

  5. Allele-Selective Transcriptome Recruitment to Polysomes Primed for Translation: Protein-Coding and Noncoding RNAs, and RNA Isoforms.

    Directory of Open Access Journals (Sweden)

    Roshan Mascarenhas

    Full Text Available mRNA translation into proteins is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs, and genetic variants remains poorly understood. mRNA levels on polysomes have been shown to correlate well with expressed protein levels, pointing to polysomal loading as a critical factor. To study regulation and genetic factors of protein translation we measured levels and allelic ratios of mRNAs and ncRNAs (including microRNAs in lymphoblast cell lines (LCL and in polysomal fractions. We first used targeted assays to measure polysomal loading of mRNA alleles, confirming reported genetic effects on translation of OPRM1 and NAT1, and detecting no effect of rs1045642 (3435C>T in ABCB1 (MDR1 on polysomal loading while supporting previous results showing increased mRNA turnover of the 3435T allele. Use of high-throughput sequencing of complete transcript profiles (RNA-Seq in three LCLs revealed significant differences in polysomal loading of individual RNA classes and isoforms. Correlated polysomal distribution between protein-coding and non-coding RNAs suggests interactions between them. Allele-selective polysome recruitment revealed strong genetic influence for multiple RNAs, attributable either to differential expression of RNA isoforms or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Genes identified by different allelic RNA ratios between cytosol and polysomes were enriched with published expression quantitative trait loci (eQTLs affecting RNA functions, and associations with clinical phenotypes. Polysomal RNA-Seq combined with allelic ratio analysis provides a powerful approach to study polysomal RNA recruitment and regulatory variants affecting protein translation.

  6. Selective sodium intercalation into sodium nickel-manganese sulfate for dual Na-Li-ion batteries.

    Science.gov (United States)

    Marinova, Delyana M; Kukeva, Rosica R; Zhecheva, Ekaterina N; Stoyanova, Radostina K

    2018-04-26

    Double sodium transition metal sulfates combine in themselves unique intercalation properties with eco-compatible compositions - a specific feature that makes them attractive electrode materials for lithium and sodium ion batteries. Herein, we examine the intercalation properties of novel double sodium nickel-manganese sulfate, Na2Ni1/2Mn1/2(SO4)2, having a large monoclinic unit cell, through electrochemical and ex situ diffraction and spectroscopic methods. The sulfate salt Na2Ni1/2Mn1/2(SO4)2 is prepared by thermal dehydration of the corresponding hydrate salt Na2Ni1/2Mn1/2(SO4)2·4H2O having a blödite structure. The intercalation reactions on Na2Ni1-xMnx(SO4)2 are studied in two model cells: half-ion cell versus Li metal anode and full-ion cell versus Li4Ti5O12 anode by using lithium (LiPF6 dissolved in EC/DMC) and sodium electrolytes (NaPF6 dissolved in EC:DEC). Based on ex situ XRD and TEM analysis, it is found that sodium intercalation into Na2Ni1/2Mn1/2(SO4)2 takes place via phase separation into the Ni-rich monoclinic phase and Mn-rich alluaudite phase. The redox reactions involving participation of manganese and titanium ions are monitored by ex situ EPR spectroscopy. It has been demonstrated that manganese ions from the sulfate salt are participating in the electrochemical reaction, while the nickel ions remain intact. As a result, a reversible capacity of about 65 mA h g-1 is reached. The selective intercalation properties determine sodium nickel-manganese sulfate as a new electrode material for hybrid lithium-sodium ion batteries that is thought to combine the advantages of individual lithium and sodium batteries.

  7. Copper, Aluminum and Nickel: A New Monocrystalline Orthodontic Alloy

    Science.gov (United States)

    Wierenga, Mark

    Introduction: This study was designed to evaluate, via tensile and bend testing, the mechanical properties of a newly-developed monocrystalline orthodontic archwire comprised of a blend of copper, aluminum, and nickel (CuAlNi). Methods: The sample was comprised of three shape memory alloys; CuAlNi, copper nickel titanium (CuNiTi), and nickel titanium (NiTi); from various orthodontic manufacturers in both 0.018" round and 0.019" x 0.025" rectangular dimensions. Additional data was gathered for similarly sized stainless steel and beta-titanium archwires as a point of reference for drawing conclusions about the relative properties of the archwires. Measurements of loading and unloading forces were recorded in both tension and deflection testing. Repeated-measure ANOVA (alpha= 0.05) was used to compare loading and unloading forces across wires and one-way ANOVA (alpha= 0.05) was used to compare elastic moduli and hysteresis. To identify significant differences, Tukey post-hoc comparisons were performed. Results: The modulus of elasticity, deflection forces, and hysteresis profiles of CuAlNi were significantly different than the other superelastic wires tested. In all tests, CuAlNi had a statistically significant lower modulus of elasticity compared to the CuNiTi and NiTi wires (P orthodontic metallurgy.

  8. Scaffold hopping from (5-hydroxymethyl) isophthalates to multisubstituted pyrimidines diminishes binding affinity to the C1 domain of protein kinase C.

    Science.gov (United States)

    Provenzani, Riccardo; Tarvainen, Ilari; Brandoli, Giulia; Lempinen, Antti; Artes, Sanna; Turku, Ainoleena; Jäntti, Maria Helena; Talman, Virpi; Yli-Kauhaluoma, Jari; Tuominen, Raimo K; Boije Af Gennäs, Gustav

    2018-01-01

    Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain-targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC.

  9. The Competitive Influences of Perceptual Load and Working Memory Guidance on Selective Attention.

    Directory of Open Access Journals (Sweden)

    Jinfeng Tan

    Full Text Available The perceptual load theory in selective attention literature proposes that the interference from task-irrelevant distractor is eliminated when perceptual capacity is fully consumed by task-relevant information. However, the biased competition model suggests that the contents of working memory (WM can guide attentional selection automatically, even when this guidance is detrimental to visual search. An intriguing but unsolved question is what will happen when selective attention is influenced by both perceptual load and WM guidance. To study this issue, behavioral performances and event-related potentials (ERPs were recorded when participants were presented with a cue to either identify or hold in memory and had to perform a visual search task subsequently, under conditions of low or high perceptual load. Behavioural data showed that high perceptual load eliminated the attentional capture by WM. The ERP results revealed an obvious WM guidance effect in P1 component with invalid trials eliciting larger P1 than neutral trials, regardless of the level of perceptual load. The interaction between perceptual load and WM guidance was significant for the posterior N1 component. The memory guidance effect on N1 was eliminated by high perceptual load. Standardized Low Resolution Electrical Tomography Analysis (sLORETA showed that the WM guidance effect and the perceptual load effect on attention can be localized into the occipital area and parietal lobe, respectively. Merely identifying the cue produced no effect on the P1 or N1 component. These results suggest that in selective attention, the information held in WM could capture attention at the early stage of visual processing in the occipital cortex. Interestingly, this initial capture of attention by WM could be modulated by the level of perceptual load and the parietal lobe mediates target selection at the discrimination stage.

  10. The Competitive Influences of Perceptual Load and Working Memory Guidance on Selective Attention.

    Science.gov (United States)

    Tan, Jinfeng; Zhao, Yuanfang; Wang, Lijun; Tian, Xia; Cui, Yan; Yang, Qian; Pan, Weigang; Zhao, Xiaoyue; Chen, Antao

    2015-01-01

    The perceptual load theory in selective attention literature proposes that the interference from task-irrelevant distractor is eliminated when perceptual capacity is fully consumed by task-relevant information. However, the biased competition model suggests that the contents of working memory (WM) can guide attentional selection automatically, even when this guidance is detrimental to visual search. An intriguing but unsolved question is what will happen when selective attention is influenced by both perceptual load and WM guidance. To study this issue, behavioral performances and event-related potentials (ERPs) were recorded when participants were presented with a cue to either identify or hold in memory and had to perform a visual search task subsequently, under conditions of low or high perceptual load. Behavioural data showed that high perceptual load eliminated the attentional capture by WM. The ERP results revealed an obvious WM guidance effect in P1 component with invalid trials eliciting larger P1 than neutral trials, regardless of the level of perceptual load. The interaction between perceptual load and WM guidance was significant for the posterior N1 component. The memory guidance effect on N1 was eliminated by high perceptual load. Standardized Low Resolution Electrical Tomography Analysis (sLORETA) showed that the WM guidance effect and the perceptual load effect on attention can be localized into the occipital area and parietal lobe, respectively. Merely identifying the cue produced no effect on the P1 or N1 component. These results suggest that in selective attention, the information held in WM could capture attention at the early stage of visual processing in the occipital cortex. Interestingly, this initial capture of attention by WM could be modulated by the level of perceptual load and the parietal lobe mediates target selection at the discrimination stage.

  11. Synthesis of new isoxazoline-based acidic amino acids and investigation of their affinity and selectivity profile at ionotropic glutamate receptors

    DEFF Research Database (Denmark)

    Pinto, Andrea; Conti, Paola; Grazioso, Giovanni

    2011-01-01

    The synthesis of four new isoxazoline-based amino acids being analogues of previously described glutamate receptor ligands is reported and their affinity for ionotropic glutamate receptors is analyzed in comparison with that of selected model compounds. Molecular modelling investigations have been...

  12. Affinity monolith chromatography: A review of principles and recent analytical applications

    Science.gov (United States)

    Pfaunmiller, Erika L.; Paulemond, Marie Laura; Dupper, Courtney M.; Hage, David S.

    2012-01-01

    Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically-related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis or studies of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments or applications of this method, with particular emphasis being given to work that has appeared in the last five years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal-ions and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing and biotechnology. Current trends and possible future directions in AMC are also discussed. PMID:23187827

  13. Positive Selection and Centrality in the Yeast and Fly Protein-Protein Interaction Networks

    Directory of Open Access Journals (Sweden)

    Sandip Chakraborty

    2016-01-01

    Full Text Available Proteins within a molecular network are expected to be subject to different selective pressures depending on their relative hierarchical positions. However, it is not obvious what genes within a network should be more likely to evolve under positive selection. On one hand, only mutations at genes with a relatively high degree of control over adaptive phenotypes (such as those encoding highly connected proteins are expected to be “seen” by natural selection. On the other hand, a high degree of pleiotropy at these genes is expected to hinder adaptation. Previous analyses of the human protein-protein interaction network have shown that genes under long-term, recurrent positive selection (as inferred from interspecific comparisons tend to act at the periphery of the network. It is unknown, however, whether these trends apply to other organisms. Here, we show that long-term positive selection has preferentially targeted the periphery of the yeast interactome. Conversely, in flies, genes under positive selection encode significantly more connected and central proteins. These observations are not due to covariation of genes’ adaptability and centrality with confounding factors. Therefore, the distribution of proteins encoded by genes under recurrent positive selection across protein-protein interaction networks varies from one species to another.

  14. Selective nickel-catalyzed conversion of model and lignin-derived phenolic compounds to cyclohexanone-based polymer building blocks.

    Science.gov (United States)

    Schutyser, Wouter; Van den Bosch, Sander; Dijkmans, Jan; Turner, Stuart; Meledina, Maria; Van Tendeloo, Gustaaf; Debecker, Damien P; Sels, Bert F

    2015-05-22

    Valorization of lignin is essential for the economics of future lignocellulosic biorefineries. Lignin is converted into novel polymer building blocks through four steps: catalytic hydroprocessing of softwood to form 4-alkylguaiacols, their conversion into 4-alkylcyclohexanols, followed by dehydrogenation to form cyclohexanones, and Baeyer-Villiger oxidation to give caprolactones. The formation of alkylated cyclohexanols is one of the most difficult steps in the series. A liquid-phase process in the presence of nickel on CeO2 or ZrO2 catalysts is demonstrated herein to give the highest cyclohexanol yields. The catalytic reaction with 4-alkylguaiacols follows two parallel pathways with comparable rates: 1) ring hydrogenation with the formation of the corresponding alkylated 2-methoxycyclohexanol, and 2) demethoxylation to form 4-alkylphenol. Although subsequent phenol to cyclohexanol conversion is fast, the rate is limited for the removal of the methoxy group from 2-methoxycyclohexanol. Overall, this last reaction is the rate-limiting step and requires a sufficient temperature (>250 °C) to overcome the energy barrier. Substrate reactivity (with respect to the type of alkyl chain) and details of the catalyst properties (nickel loading and nickel particle size) on the reaction rates are reported in detail for the Ni/CeO2 catalyst. The best Ni/CeO2 catalyst reaches 4-alkylcyclohexanol yields over 80 %, is even able to convert real softwood-derived guaiacol mixtures and can be reused in subsequent experiments. A proof of principle of the projected cascade conversion of lignocellulose feedstock entirely into caprolactone is demonstrated by using Cu/ZrO2 for the dehydrogenation step to produce the resultant cyclohexanones (≈80 %) and tin-containing beta zeolite to form 4-alkyl-ε-caprolactones in high yields, according to a Baeyer-Villiger-type oxidation with H2 O2 . © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Identification of Protein Complexes from Tandem Affinity Purification/Mass Spectrometry Data via Biased Random Walk.

    Science.gov (United States)

    Cai, Bingjing; Wang, Haiying; Zheng, Huiru; Wang, Hui

    2015-01-01

    Systematic identification of protein complexes from protein-protein interaction networks (PPIs) is an important application of data mining in life science. Over the past decades, various new clustering techniques have been developed based on modelling PPIs as binary relations. Non-binary information of co-complex relations (prey/bait) in PPIs data derived from tandem affinity purification/mass spectrometry (TAP-MS) experiments has been unfairly disregarded. In this paper, we propose a Biased Random Walk based algorithm for detecting protein complexes from TAP-MS data, resulting in the random walk with restarting baits (RWRB). RWRB is developed based on Random walk with restart. The main contribution of RWRB is the incorporation of co-complex relations in TAP-MS PPI networks into the clustering process, by implementing a new restarting strategy during the process of random walk. Through experimentation on un-weighted and weighted TAP-MS data sets, we validated biological significance of our results by mapping them to manually curated complexes. Results showed that, by incorporating non-binary, co-membership information, significant improvement has been achieved in terms of both statistical measurements and biological relevance. Better accuracy demonstrates that the proposed method outperformed several state-of-the-art clustering algorithms for the detection of protein complexes in TAP-MS data.

  16. The affinity purification and characterization of ATP synthase complexes from mitochondria.

    Science.gov (United States)

    Runswick, Michael J; Bason, John V; Montgomery, Martin G; Robinson, Graham C; Fearnley, Ian M; Walker, John E

    2013-02-13

    The mitochondrial F₁-ATPase inhibitor protein, IF₁, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF₁ occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF₁ with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1-60 of bovine IF₁ with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.

  17. Process for forming a nickel foil with controlled and predetermined permeability to hydrogen

    Science.gov (United States)

    Engelhaupt, Darell E.

    1981-09-22

    The present invention provides a novel process for forming a nickel foil having a controlled and predetermined hydrogen permeability. This process includes the steps of passing a nickel plating bath through a suitable cation exchange resin to provide a purified nickel plating bath free of copper and gold cations, immersing a nickel anode and a suitable cathode in the purified nickel plating bath containing a selected concentration of an organic sulfonic acid such as a napthalene-trisulfonic acid, electrodepositing a nickel layer having the thickness of a foil onto the cathode, and separating the nickel layer from the cathode to provide a nickel foil. The anode is a readily-corrodible nickel anode. The present invention also provides a novel nickel foil having a greater hydrogen permeability than palladium at room temperature.

  18. Process for forming a nickel foil with controlled and predetermined permeability to hydrogen

    International Nuclear Information System (INIS)

    Engelhaupt, D. E.

    1981-01-01

    The present invention provides a novel process for forming a nickel foil having a controlled and predetermined hydrogen permeability. This process includes the steps of passing a nickel plating bath through a suitable cation exchange resin to provide a purified nickel plating bath free of copper and gold cations, immersing a nickel anode and a suitable cathode in the purified nickel plating bath containing a selected concentration of an organic sulfonic acid such as a napthalene-trisulfonic acid, electrodepositing a nickel layer having the thickness of a foil onto the cathode, and separating the nickel layer from the cathode to provide a nickel foil. The anode is a readilycorrodible nickel anode. The present invention also provides a novel nickel foil having a greater hydrogen permeability than palladium at room temperature

  19. Antibody Affinity Maturation in Fishes—Our Current Understanding

    Directory of Open Access Journals (Sweden)

    Brad G. Magor

    2015-07-01

    Full Text Available It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig mutator enzyme activation-induced cytidine deaminase (AID. We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes.

  20. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi-Fen; Shyu, Huey-Wen [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chang, Yi-Chuang [Department of Nursing, Fooyin University, Kaohsiung, Taiwan (China); Tseng, Wei-Chang [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Huang, Yeou-Lih [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chen, Chang-Yu, E-mail: mt037@mail.fy.edu.tw [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China)

    2012-03-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  1. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    International Nuclear Information System (INIS)

    Wang, Yi-Fen; Shyu, Huey-Wen; Chang, Yi-Chuang; Tseng, Wei-Chang; Huang, Yeou-Lih; Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling; Chen, Chang-Yu

    2012-01-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  2. Absolute determination by X-ray diffraction of a binary or ternary mixture: nickel oxide and fluoride in a nickel powder (1960)

    International Nuclear Information System (INIS)

    Charpin, P.; Hauptman, A.

    1960-01-01

    The method employed is based upon the comparison between computed and measured intensities for conveniently selected X-Ray diffraction lines of each component of the powder. Care must be taken to allow for absorption, both inside each grain and in overall sample. This method has been applied to the determination of nickel oxide and fluoride in a nickel powder. (author) [fr

  3. Different endothelin receptor affinities in dog tissues

    International Nuclear Information System (INIS)

    Loeffler, B.M.L.; Loehrer, W.

    1991-01-01

    Endothelin (ET) is a long-lasting potent vasoconstrictor-peptide. Here the authors report different binding affinities of endothelin-1 (ET-1) to ET-receptors of various dog tissues. Crude microsomal fractions were prepared after homogenisation of dog tissues in 50 mM Tris/HCl, 20 mM MnCl2, 1 mM EDTA, pH 7.4 by differential centrifugation. Aliquots of microsomal fractions (70 micrograms of protein) were incubated at 25 degrees C for 180 min in the presence of 20 pM 125I-ET-1 and various concentrations of cold ET-1. Four different ET-1 receptor binding affinities were found: adrenals, cerebrum, liver, heart, skeletal muscle and stomach microsomal membranes contained high affinity binding sites (Kd 50 - 80 pM, Bmax 60 - 250 fmol/mg). In cerebellum and spleen medium affinity ET-1 receptors (Kd 350 pM, Bmax 880 and 1200 fmol/mg respectively) were present. In comparison lung and kidney microsomes contained a low affinity ET-1 receptor (Kd 800 and 880 pM, Bmax 1600 and 350 fmol/mg). Receptors of even lower affinity were present in heart, intestine and liver microsomes with Kd values of 3 - 6 nM

  4. Nickel-induced cytokine production from mononuclear cells in nickel-sensitive individuals and controls. Cytokine profiles in nickel-sensitive individuals with nickel allergy-related hand eczema before and after nickel challenge

    DEFF Research Database (Denmark)

    Borg, L; Christensen, J M; Kristiansen, J

    2000-01-01

    Exposure to nickel is a major cause of allergic contact dermatitis which is considered to be an inflammatory response induced by antigen-specific T cells. Here we describe the in vitro analysis of the nickel-specific T-cell-derived cytokine response of peripheral blood mononuclear cells from 35...... was somewhat of a surprise, since previous studies have suggested a Th1 response in nickel-mediated allergic contact dermatitis. Subsequently, the nickel-allergic individuals were randomized to experimental exposure to nickel or vehicle in a double-blind design. A daily 10-min exposure of one finger to 10 ppm...... nickel solution for 1 week followed by 100 ppm for an additional week evoked a clinical response of hand eczema in the nickel-exposed group. Blood samples were drawn on days 7 and 14 after the start of this exposure to occupationally relevant concentrations of nickel. No statistically significant...

  5. IA-2 autoantibody affinity in children at risk for type 1 diabetes.

    Science.gov (United States)

    Krause, Stephanie; Chmiel, Ruth; Bonifacio, Ezio; Scholz, Marlon; Powell, Michael; Furmaniak, Jadwiga; Rees Smith, Bernard; Ziegler, Anette-G; Achenbach, Peter

    2012-12-01

    Autoantibodies to insulinoma-associated protein 2 (IA-2A) are associated with increased risk for type 1 diabetes. Here we examined IA-2A affinity and epitope specificity to assess heterogeneity in response intensity in relation to pathogenesis and diabetes risk in 50 children who were prospectively followed from birth. At first IA-2A appearance, affinity ranged from 10(7) to 10(11)L/mol and was high (>1.0×10(9)L/mol) in 41 (82%) children. IA-2A affinity was not associated with epitope specificity or HLA class II haplotype. On follow-up, affinity increased or remained high, and IA-2A were commonly against epitopes within the protein tyrosine phosphatase-like IA-2 domain and the homologue protein IA-2β. IA-2A were preceded or accompanied by other islet autoantibodies in 49 (98%) children, of which 34 progressed to diabetes. IA-2A affinity did not stratify diabetes risk. In conclusion, the IA-2A response in children is intense with rapid maturation against immunogenic epitopes and a strong association with diabetes development. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. A rapid method for selecting suitable animal species for studying pathogen interactions with plasma protein ligands in vivo.

    Science.gov (United States)

    Naudin, Clément; Schumski, Ariane; Salo-Ahen, Outi M H; Herwald, Heiko; Smeds, Emanuel

    2017-05-01

    Species tropism constitutes a serious problem for developing relevant animal models of infection. Human pathogens can express virulence factors that show specific selectivity to human proteins, while their affinity for orthologs from other species can vary significantly. Suitable animal species must be used to analyse whether virulence factors are potential targets for drug development. We developed an assay that rapidly predicts applicable animal species for studying virulence factors binding plasma proteins. We used two well-characterized Staphylococcus aureus proteins, SSL7 and Efb, to develop an ELISA-based inhibition assay using plasma from different animal species. The interaction between SSL7 and human C5 and the binding of Efb to human fibrinogen and human C3 was studied. Affinity experiments and Western blot analyses were used to validate the assay. Human, monkey and cat plasma interfered with binding of SSL7 to human C5. Binding of Efb to human fibrinogen was blocked in human, monkey, gerbil and pig plasma, while human, monkey, gerbil, rabbit, cat and guinea pig plasma inhibited the binding of Efb to human C3. These results emphasize the importance of choosing correct animal models, and thus, our approach is a rapid and cost-effective method that can be used to prevent unnecessary animal experiments. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  7. Weak affinity chromatography for evaluation of stereoisomers in early drug discovery.

    Science.gov (United States)

    Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Svensson, Susanne; Ohlson, Sten; Isaksson, Roland

    2013-07-01

    In early drug discovery (e.g., in fragment screening), recognition of stereoisomeric structures is valuable and guides medicinal chemists to focus only on useful configurations. In this work, we concurrently screened mixtures of stereoisomers and estimated their affinities to a protein target (thrombin) using weak affinity chromatography-mass spectrometry (WAC-MS). Affinity determinations by WAC showed that minor changes in stereoisomeric configuration could have a major impact on affinity. The ability of WAC-MS to provide instant information about stereoselectivity and binding affinities directly from analyte mixtures is a great advantage in fragment library screening and drug lead development.

  8. A Versatile Platform to Analyze Low-Affinity and Transient Protein-Protein Interactions in Living Cells in Real Time

    Directory of Open Access Journals (Sweden)

    Yao-Cheng Li

    2014-12-01

    Full Text Available Summary: Protein-protein interactions (PPIs play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL’s ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL’s ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms. : Li et al. developed a recombinase-enhanced bimolecular luciferase complementation platform, termed ReBiL, to evaluate low-affinity protein-protein interactions (PPIs that are not detectable by other methods and to analyze PPI antagonists in living cells. ReBiL showed that small-molecule p53-Mdm2 antagonists disrupt their intended targets effectively in cells, whereas stapled peptides did not. Stapled peptides unexpectedly induced cell membrane disruption resulting in p53-independent death associated with cytoplasmic leakage. ReBiL is also valuable for high-throughput screening and for deciphering signaling mechanisms mediated by protein interactions.

  9. Twist on protein microarrays: layering wax-patterned nitrocellulose to create customizable and separable arrays of multiplexed affinity columns.

    Science.gov (United States)

    de Lange, Victoria; Vörös, János

    2014-05-06

    We developed the simple and inexpensive FoRe microarray to simultaneously test several 1 μL samples for multiple proteins. By combining forward and reverse phase microarrays into an innovative three-dimensional format, the FoRe array exploits the advantages and eliminates several drawbacks of the traditional approaches (i.e., large sample volumes, protein loss, and cross-reactivity between detection antibodies). Samples are pipetted into an array of separable, multiplexed affinity columns. Several nitrocellulose membranes, each functionalized with a different capture antibody, are stacked to create a customizable affinity column. The nitrocellulose is patterned with wax to form 25 isolated microspots on each layer, allowing us to analyze multiple samples in parallel. After running the immunoassay, the stacks are quickly disassembled, revealing 2D microarrays of different fractions from multiple samples. By combining the stack-and-separate technique with wax patterning, we keep the arrays low cost and easily tailored to a variety of applications. We successfully performed 3D multiplexing using a model system with mouse and rabbit IgG. Binding proved to be independent of the position in the stack, and the limit of detection for a mouse IgG sandwich assay was 7.3 pM in BSA and 15 pM in human plasma. The FoRe microarray makes it possible to identify protein expression patterns across several minute volume samples; for example, it could be used to analyze cell lysate in drug response studies or pricks of blood from small animal studies.

  10. Selection of peptides interfering with protein-protein interaction.

    Science.gov (United States)

    Gaida, Annette; Hagemann, Urs B; Mattay, Dinah; Räuber, Christina; Müller, Kristian M; Arndt, Katja M

    2009-01-01

    Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided interactions are often associated with various diseases. Consequently, peptides, which are able to specifically interfere with such adventitious interactions, are of high interest for analytical as well as medical purposes. One of the most abundant protein interaction domains is the coiled-coil motif, and thus provides a premier target. Coiled coils, which consist of two or more alpha-helices wrapped around each other, have one of the simplest interaction interfaces, yet they are able to confer highly specific homo- and heterotypic interactions involved in virtually any cellular process. While there are several ways to generate interfering peptides, the combination of library design with a powerful selection system seems to be one of the most effective and promising approaches. This chapter guides through all steps of such a process, starting with library options and cloning, detailing suitable selection techniques and ending with purification for further down-stream characterization. Such generated peptides will function as versatile tools to interfere with the natural function of their targets thereby illuminating their down-stream signaling and, in general, promoting understanding of factors leading to specificity and stability in protein-protein interactions. Furthermore, peptides interfering with medically relevant proteins might become important diagnostics and therapeutics.

  11. Heme-binding plasma membrane proteins of K562 erythroleukemia cells: Adsorption to heme-microbeads, isolation with affinity chromatography

    International Nuclear Information System (INIS)

    Majuri, R.

    1989-01-01

    Heme-microbeads attached themselves to the surface of viable K562 cells in a manner inhibitable by free hemin, indicating heme-recptor interaction. The microbeads were at first evenly distributed, but after prolonged incubation at 37 deg. C they formed a cap on one pole of the cells indicating clustering of the membrane heme receptors. Membrane proteins were labeled by culturing the cells in the presence of 35 S-methionine and were then solubilized with Triton X-114. The hydrophobic proteins contained about 20% of the total bound label. The solubilized membrane proteins were subsequently adsorbed to a heme-Sepharose affinity gel. According to SDS-electrophorsis and subsequent autoradiography, the immobilized heme captures two proteins or a protein with two polypeptides of 20 000 and 32 000 daltons. The larger of these was only wekly labeled with 35 S. The same two bands were observed if the cell surface proteins were labeled with 125 I by the lactoperoxidase method and the subsequently solubilized membrane proteins were isolated with heme-Sepharose. (author)

  12. Mutational analysis of affinity and selectivity of kringle-tetranectin interaction. Grafting novel kringle affinity ontp the trtranectin lectin scaffold

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Jacobsen, C; Sigurskjold, B W

    2000-01-01

    -type lectin-like domain of tetranectin, involving Lys-148, Glu-150, and Asp-165, which mediates calcium-sensitive binding to plasminogen kringle 4. Here, we investigate the effect of conservative substitutions of these and a neighboring amino acid residue. Substitution of Thr-149 in tetranectin...... with a tyrosine residue considerably increases the affinity for plasminogen kringle 4, and, in addition, confers affinity for plasminogen kringle 2. As shown by isothermal titration calorimetry analysis, this new interaction is stronger than the binding of wild-type tetranectin to plasminogen kringle 4...

  13. A single acidic residue can guide binding site selection but does not govern QacR cationic-drug affinity.

    Directory of Open Access Journals (Sweden)

    Kate M Peters

    Full Text Available Structures of the multidrug-binding repressor protein QacR with monovalent and bivalent cationic drugs revealed that the carboxylate side-chains of E90 and E120 were proximal to the positively charged nitrogens of the ligands ethidium, malachite green and rhodamine 6G, and therefore may contribute to drug neutralization and binding affinity. Here, we report structural, biochemical and in vivo effects of substituting these glutamate residues. Unexpectedly, substitutions had little impact on ligand affinity or in vivo induction capabilities. Structures of QacR(E90Q and QacR(E120Q with ethidium or malachite green took similar global conformations that differed significantly from all previously described QacR-drug complexes but still prohibited binding to cognate DNA. Strikingly, the QacR(E90Q-rhodamine 6G complex revealed two mutually exclusive rhodamine 6G binding sites. Despite multiple structural changes, all drug binding was essentially isoenergetic. Thus, these data strongly suggest that rather than contributing significantly to ligand binding affinity, the role of acidic residues lining the QacR multidrug-binding pocket is primarily to attract and guide cationic drugs to the "best available" positions within the pocket that elicit QacR induction.

  14. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen

    2012-01-01

    Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

  15. Nickel adsorption and desorption in an acric oxisol as a function of pH, ionic strength and incubation time

    Directory of Open Access Journals (Sweden)

    Estêvão Vicari Mellis

    Full Text Available ABSTRACT Although nickel (Ni has both important potential benefits and toxic effects in the environment, its behavior in tropical soils has not been well studied. Nickel adsorption-desorption in topsoil and subsoil samples of an acric Oxisol was studied at three pH values (from 3.0 to 8.0. Adsorption-desorption isotherms were elaborated from experiments with increasing Ni concentration (5 to 100 mg L-1, during 0, 4, and 12 weeks, using CaCl2 0.01 and 0.1 M as electrolytic support in order to also verify the effect of Ni-soil time contact and of ionic strength on the reaction. Experimental results of Ni adsorption fitted Langmuir model, which indicated that maximum Ni adsorption (71,440 mg kg-1 occurred at subsoil, after 12 weeks. Nickel affinity (KL was also greater at subsoil (1.0 L kg-1. The Ni adsorption in the topsoil samples was higher, due to its lower point of zero salt effect (PZSE and higher organic matter content. The increase in soil pH resulted in the increase of Ni adsorption. Nickel desorbed less from soil samples incubated for 4 or 12 weeks, suggesting that Ni interactions with colloidal particles increase over time. The amount of Ni desorbed increased with increasing ionic strength in both the topsoil and subsoil soil samples. Finally, adsorption-desorption hysteresis was clearly observed. Soil pH, ionic strength of soil solution and the Ni-soil contact time should be considered as criteria for selecting the areas for disposal of residues containing Ni or to compose remediation strategies for acric soils contaminated with Ni.

  16. Capped Mesoporous Silica Nanoparticles for the Selective and Sensitive Detection of Cyanide.

    Science.gov (United States)

    Sayed, Sameh El; Licchelli, Maurizio; Martínez-Máñez, Ramón; Sancenón, Félix

    2017-10-18

    The development of easy and affordable methods for the detection of cyanide is of great significance due to the high toxicity of this anion and the potential risks associated with its pollution. Herein, optical detection of cyanide in water has been achieved by using a hybrid organic-inorganic nanomaterial. Mesoporous silica nanoparticles were loaded with [Ru(bipy) 3 ] 2+ , functionalized with macrocyclic nickel(II) complex subunits, and capped with a sterically hindering anion (hexametaphosphate). Cyanide selectively induces demetallation of nickel(II) complexes and the removal of capping anions from the silica surface, allowing the release of the dye and the consequent increase in fluorescence intensity. The response of the capped nanoparticles in aqueous solution is highly selective and sensitive towards cyanide with a limit of detection of 2 μm. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. The neural correlates of perceptual load induced attentional selection: an fMRI study.

    Science.gov (United States)

    Wei, P; Szameitat, A J; Müller, H J; Schubert, T; Zhou, X

    2013-10-10

    The neural correlates of perceptual load induced attentional selection were investigated in an functional magnetic resonance imaging (fMRI) experiment in which attentional selection was manipulated through the variation of perceptual load in target search. Participants searched for a vertically or horizontally oriented bar among heterogeneously (the high load condition) or homogeneously (the low load condition) oriented distractor bars in the central display, which was flanked by a vertical or horizontal bar presented at the left or the right periphery. The search reaction times were longer when the central display was of high load than of low load, and were longer when the flanker was incongruent than congruent with the target. Importantly, the flanker congruency effect was manifested only in the low load condition, not in the high load condition, indicating that the perceptual load in target search determined whether the task-irrelevant flanker was processed. Imaging analyses revealed a set of fronto-parietal regions having higher activations in the high than in the low load condition. Anterior cingulate cortex (ACC) was more activated for the incongruent than for the congruent trials. Moreover, ACC and bilateral anterior insula were sensitive to the interaction between perceptual load and flanker congruency such that the activation differences between the incongruent and congruent conditions were significant in the low, but not in the high load condition. These results are consistent with the claim that ACC and bilateral anterior insula may exert executive control by selectively biasing processing in favor of task-relevant information and this biasing depends on the resources currently available to the control system. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  18. Tandem affinity purification of histones, coupled to mass spectrometry, identifies associated proteins and new sites of post-translational modification in Saccharomyces cerevisiae.

    Science.gov (United States)

    Valero, M Luz; Sendra, Ramon; Pamblanco, Mercè

    2016-03-16

    Histones and their post-translational modifications contribute to regulating fundamental biological processes in all eukaryotic cells. We have applied a conventional tandem affinity purification strategy to histones H3 and H4 of the yeast Saccharomyces cerevisiae. Mass spectrometry analysis of the co-purified proteins revealed multiple associated proteins, including core histones, which indicates that tagged histones may be incorporated to the nucleosome particle. Among the many other co-isolated proteins there are histone chaperones, elements of chromatin remodeling, of nucleosome assembly/disassembly, and of histone modification complexes. The histone chaperone Rtt106p, two members of chromatin assembly FACT complex and Psh1p, an ubiquitin ligase, were the most abundant proteins obtained with both H3-TAP and H4-TAP, regardless of the cell extraction medium stringency. Our mass spectrometry analyses have also revealed numerous novel post-translational modifications, including 30 new chemical modifications in histones, mainly by ubiquitination. We have discovered not only new sites of ubiquitination but that, besides lysine, also serine and threonine residues are targets of ubiquitination on yeast histones. Our results show the standard tandem affinity purification procedure is suitable for application to yeast histones, in order to isolate and characterize histone-binding proteins and post-translational modifications, avoiding the bias caused by histone purification from a chromatin-enriched fraction. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Nickel-titanium alloys: stress-related temperature transitional range.

    Science.gov (United States)

    Santoro, M; Beshers, D N

    2000-12-01

    The inducement of mechanical stress within nickel-titanium wires can influence the transitional temperature range of the alloy and therefore the expression of the superelastic properties. An analogous variation of the transitional temperature range may be expected during orthodontic therapy, when the archwires are engaged into the brackets. To investigate this possibility, samples of currently used orthodontic nickel-titanium wires (Sentalloy, GAC; Copper Ni-Ti superelastic at 27 degrees C, 35 degrees C, 40 degrees C, Ormco; Nitinol Heat-Activated, 3M-Unitek) were subjected to temperature cycles ranging between 4 degrees C and 60 degrees C. The wires were mounted in a plexiglass loading device designed to simulate clinical situations of minimum and severe dental crowding. Electrical resistivity was used to monitor the phase transformations. The data were analyzed with paired t tests. The results confirmed the presence of displacements of the transitional temperature ranges toward higher temperatures when stress was induced. Because nickel-titanium wires are most commonly used during the aligning stage in cases of severe dental crowding, particular attention was given to the performance of the orthodontic wires under maximum loading. An alloy with a stress-related transitional temperature range corresponding to the fluctuations of the oral temperature should express superelastic properties more consistently than others. According to our results, Copper Ni-Ti 27 degrees C and Nitinol Heat-Activated wires may be considered suitable alloys for the alignment stage.

  20. Accurate and Reliable Prediction of the Binding Affinities of Macrocycles to Their Protein Targets.

    Science.gov (United States)

    Yu, Haoyu S; Deng, Yuqing; Wu, Yujie; Sindhikara, Dan; Rask, Amy R; Kimura, Takayuki; Abel, Robert; Wang, Lingle

    2017-12-12

    Macrocycles have been emerging as a very important drug class in the past few decades largely due to their expanded chemical diversity benefiting from advances in synthetic methods. Macrocyclization has been recognized as an effective way to restrict the conformational space of acyclic small molecule inhibitors with the hope of improving potency, selectivity, and metabolic stability. Because of their relatively larger size as compared to typical small molecule drugs and the complexity of the structures, efficient sampling of the accessible macrocycle conformational space and accurate prediction of their binding affinities to their target protein receptors poses a great challenge of central importance in computational macrocycle drug design. In this article, we present a novel method for relative binding free energy calculations between macrocycles with different ring sizes and between the macrocycles and their corresponding acyclic counterparts. We have applied the method to seven pharmaceutically interesting data sets taken from recent drug discovery projects including 33 macrocyclic ligands covering a diverse chemical space. The predicted binding free energies are in good agreement with experimental data with an overall root-mean-square error (RMSE) of 0.94 kcal/mol. This is to our knowledge the first time where the free energy of the macrocyclization of linear molecules has been directly calculated with rigorous physics-based free energy calculation methods, and we anticipate the outstanding accuracy demonstrated here across a broad range of target classes may have significant implications for macrocycle drug discovery.

  1. Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

    International Nuclear Information System (INIS)

    Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

    2008-01-01

    In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 ± 0.7 x 10 5 M -1 which indicates a strong binding close to that of antibody

  2. Working memory load can both improve and impair selective attention: evidence from the Navon paradigm.

    Science.gov (United States)

    Ahmed, Lubna; de Fockert, Jan W

    2012-10-01

    Selective attention to relevant targets has been shown to depend on the availability of working memory (WM). Under conditions of high WM load, processing of irrelevant distractors is enhanced. Here we showed that this detrimental effect of WM load on selective attention efficiency is reversed when the task requires global- rather than local-level processing. Participants were asked to attend to either the local or the global level of a hierarchical Navon stimulus while keeping either a low or a high load in WM. In line with previous findings, during attention to the local level, distractors at the global level produced more interference under high than under low WM load. By contrast, loading WM had the opposite effect of improving selective attention during attention to the global level. The findings demonstrate that the impact of WM load on selective attention is not invariant, but rather is dependent on the level of the to-be-attended information.

  3. Affinity purification of recombinant human plasminogen activator ...

    African Journals Online (AJOL)

    Affinity purification of recombinant human plasminogen activator from ... Screening antibody was performed using rhPA milk in an ELISA-elution assay. ... useful for purifying other tPA mutants or other novel recombinant milkderived proteins.

  4. NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

    Science.gov (United States)

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

    2002-07-01

    Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

  5. Removing nickel from nickel-coated carbon fibers

    Science.gov (United States)

    Hardianto, A.; Hertleer, C.; De Mey, G.; Van Langenhove, L.

    2017-10-01

    Conductive fibers/yarns are one of the most important materials for smart textiles because of their electrically conductive functionality combined with flexibility and light weight. They can be applied in many fields such as the medical sector, electronics, sensors and even as thermoelectric generators. Temperature sensors, for example, can be made using the thermocouple or thermopile principle which usually uses two different metal wires that can produce a temperature-dependent voltage. However, if metal wires are inserted into a textile structure, they will decrease the flexibility properties of the textile product. Nickel-coated Carbon Fiber (NiCF), a conductive textile yarn, has a potential use as a textile-based thermopile if we can create an alternating region of carbon and nickel along the fiber which in turn it can be used for substituting the metallic thermopile. The idea was to remove nickel from NiCF in order to obtain a yarn that contains alternating zones of carbon and nickel. Due to no literature reporting on how to remove nickel from NiCF, in this paper we investigated some chemicals to remove nickel from NiCF.

  6. Behavioral interventions to reduce nickel exposure in a nickel processing plant.

    Science.gov (United States)

    Rumchev, Krassi; Brown, Helen; Wheeler, Amanda; Pereira, Gavin; Spickett, Jeff

    2017-10-01

    Nickel is a widely-used material in many industries. Although there is enough evidence that occupational exposure to nickel may cause respiratory illnesses, allergies, and even cancer, it is not possible to stop the use of nickel in occupational settings. Nickel exposure, however, can be controlled and reduced significantly in workplaces. The main objective of this study was to assess if educational intervention of hygiene behavior could reduce nickel exposure among Indonesian nickel smelter workers. Participants were randomly assigned to three intervention groups (n = 99). Group one (n = 35) received only an educational booklet about nickel, related potential health effects and preventive measures, group two (n = 35) attended a presentation in addition to the booklet, and group three (n = 29) received personal feedback on their biomarker results in addition to the booklet and presentations. Pre- and post-intervention air sampling was conducted to measure concentrations of dust and nickel in air along with worker's blood and urine nickel concentrations. The study did not measure significant differences in particles and nickel concentrations in the air between pre- and post-interventions. However, we achieved significant reductions in the post intervention urine and blood nickel concentrations which can be attributed to changes in personal hygiene behavior. The median urinary nickel concentration in the pre-intervention period for group one was 52.3 µg/L, for group two 57.4 µg/L, and group three 43.2 µg/L which were significantly higher (pnickel with significantly (p nickel levels of 0.1 µg/L for all groups. The study showed that educational interventions can significantly reduce personal exposure levels to nickel among Indonesian nickel smelter workers.

  7. Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

    Czech Academy of Sciences Publication Activity Database

    Tykvart, Jan; Šácha, Pavel; Bařinka, Cyril; Knedlík, Tomáš; Starková, Jana; Lubkowski, J.; Konvalinka, Jan

    2012-01-01

    Roč. 82, č. 1 (2012), s. 106-115 ISSN 1046-5928 R&D Projects: GA MŠk 1M0508; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520701 Keywords : affinity purification * biotin acceptor peptide * recombinant protein expression * biotin -protein ligase (BirA) * co-localization * PSMA Subject RIV: CE - Biochemistry Impact factor: 1.429, year: 2012

  8. Fundamental and practical studies on high-performance liquid affinity chromatography of biopolymers with novel stationary phases

    Energy Technology Data Exchange (ETDEWEB)

    Bacolod, M.D.

    1992-01-01

    Rigid microparticulate stationary phases having surface-bound metal chelating functions were developed and evaluated in high performance metal chelate affinity chromatography of proteins. Silica- and polystyrene-divinylbenzene-based metal chelate sorbents were produced in wide pore and in non-porous type of column packings. A major effort has been placed on development of non-porous highly crosslinked polystyrene-divinylbenzene (PSDVB). These PSDVB microparticles were produced by a two-step swelling polymerization, and exhibited excellent mechanical strength over a wide range of flow-rates and composition used in high performance liquid chromatography (HPLC). Simple and reproducible hydrophilic coatings were developed for the surface modification of hydrophobic PSDVB supports. A tetradentate metal chelating ligand, ethylenediamine-N, N[prime]-diacetic acid (EDDA), was covalently bound to the surface of the various supports. Sorbents having iminodiacetic acid (IDA) metal chelating functions were also evaluated. The hydrophilic character and surface coverage of various stationary phases were assessed chromatographically. Studies concerning the effects of eluent pH as well as the nature and concentration of salts on retention and selectivity with different metal chelate stationary phases having various immobilized metal ions were carried out. Elution schemes were developed for rapid separation of proteins in metal chelate affinity chromatography. EDDA stationary phases in metal forms can be viewed as complementary to IDA stationary phases since they afforded different selectivity and retentivity toward proteins. Hydrophilic PSDVB could be functionalized with IDA or EDDA metal chelating ligands or lectins. The non-porous metal chelate stationary phases afforded rapid separation of proteins by the development of multiple gradient systems, which permitted higher column peak capacity, enabling the separation of a greater number of proteins in a single chromatographic run.

  9. Sintering of nickel catalysts. Effects of time, atmosphere, temperature, nickel-carrier interactions, and dopants

    Energy Technology Data Exchange (ETDEWEB)

    Sehested, Jens; Gelten, Johannes A.P.; Helveg, Stig [Haldor Topsoee A/S, Nymoellevej 55, DK-2800 Kgs. Lyngby (Denmark)

    2006-08-01

    Supported nickel catalysts are widely used in the steam-reforming process for industrial scale production of hydrogen and synthesis gas. This paper provides a study of sintering in nickel-based catalysts (Ni/Al{sub 2}O{sub 3} and Ni/MgAl{sub 2}O{sub 4}). Specifically the influence of time, temperature, atmosphere, nickel-carrier interactions and dopants on the rate of sintering is considered. To probe the sintering kinetics, all catalysts were analyzed by sulfur chemisorption to determine the Ni surface area. Furthermore selected samples were further analyzed using X-ray diffraction (XRD), mercury porosimetry, BET area measurements, and electron microscopy (EM). The observed sintering rates as a function of time, temperature, and P{sub H{sub 2}O}/P{sub H{sub 2}} ratio were consistent with recent model predictions [J. Sehested, J.A.P. Gelten, I.N. Remediakis, H. Bengaard, J.K. Norskov, J. Catal. 223 (2004) 432] over a broad range of environmental conditions. However, exposing the catalysts to severe sintering conditions the loss of nickel surface area is faster than model predictions and the deviation is attributed to a change in the sintering mechanism and nickel removal by nickel-carrier interactions. Surprisingly, alumina-supported Ni particles grow to sizes larger than the particle size of the carrier indicating that the pore diameter does not represent an upper limit for Ni particle growth. The effects of potassium promotion and sulfur poisoning on the rates of sintering were also investigated. No significant effects of the dopants were observed after ageing at ambient pressure. However, at high pressures of steam and hydrogen (31bar and H{sub 2}O:H{sub 2}=10:1) potassium promotion increased the sintering rate relative to that of the unpromoted catalyst. Sulfur also enhances the rate of sintering at high pressures, but the effect of sulfur is less than for potassium. (author)

  10. Affinity Electrophoresis for Analysis of Catalytic Module-Carbohydrate Interactions

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Svensson, Birte

    2017-01-01

    Affinity electrophoresis has long been used to study the interaction between proteins and large soluble ligands. The technique has been found to have great utility for the examination of polysaccharide binding by proteins, particularly carbohydrate binding modules (CBMs). In recent years, carbohy...

  11. Long Life Nickel Electrodes for Nickel-Hydrogen Cells: Fiber Substrates Nickel Electrodes

    Science.gov (United States)

    Rogers, Howard H.

    2000-01-01

    Samples of nickel fiber mat electrodes were investigated over a wide range of fiber diameters, electrode thickness, porosity and active material loading levels. Thickness' were 0.040, 0.060 and 0.080 inches for the plaque: fiber diameters were primarily 2, 4, and 8 micron and porosity was 85, 90, and 95%. Capacities of 3.5 in. diameter electrodes were determined in the flooded condition with both 26 and 31% potassium hydroxide solution. These capacity tests indicated that the highest capacities per unit weight were obtained at the 90% porosity level with a 4 micron diameter fiber plaque. It appeared that the thinner electrodes had somewhat better performance, consistent with sintered electrode history. Limited testing with two-positive-electrode boiler plate cells was also carried out. Considerable difficulty with constructing the cells was encountered with short circuits the major problem. Nevertheless, four cells were tested. The cell with 95% porosity electrodes failed during conditioning cycling due to high voltage during charge. Discharge showed that this cell had lost nearly all of its capacity. The other three cells after 20 conditioning cycles showed capacities consistent with the flooded capacities of the electrodes. Positive electrodes made from fiber substrates may well show a weight advantage of standard sintered electrodes, but need considerably more work to prove this statement. A major problem to be investigated is the lower strength of the substrate compared to standard sintered electrodes. Problems with welding of leads were significant and implications that the electrodes would expand more than sintered electrodes need to be investigated. Loading levels were lower than had been expected based on sintered electrode experiences and the lower loading led to lower capacity values. However, lower loading causes less expansion and contraction during cycling so that stress on the substrate is reduced.

  12. Chirality and grain boundary effects on indentation mechanical properties of graphene coated on nickel foil

    Science.gov (United States)

    Yan, Yuping; Lv, Jiajiang; Liu, Sheng

    2018-04-01

    We investigate chirality and grain boundary (GB) effects on indentation mechanical properties of graphene coated on nickel foil using molecular dynamics simulations. The models of graphene with different chirality angles, different numbers of layers and tilt GBs were established. It was found that the chirality angle of few-layer graphene had a significant effect on the load bearing capacity of graphene/nickel systems, and this turns out to be more significant when the number of layers is greater than one. The enhancement to the contact stiffness, elastic capacity and the load bearing capacity of graphene with tilt GBs was lower than that of pristine graphene.

  13. Proposed sale of radioactively contaminated nickel ingots located at the Paducah Gaseous Diffusion Plant, Paducah, Kentucky

    International Nuclear Information System (INIS)

    1995-10-01

    The U.S. Department of Energy (DOE) proposes to sell 8,500 radioactively contaminated nickel ingots (9.350 short tons), currently in open storage at the Paducah Gaseous Diffusion Plant (PGDP), to Scientific Ecology Group, Inc. (SEG) for decontamination and resale on the international market. SEG would take ownership of the ingots when they are loaded for transport by truck to its facility in Oak Ridge, Tennessee. SEG would receive approximately 200 short tons per month over approximately 48 months (an average of 180 ingots per month). The nickel decontamination process specified in SEG's technical proposal is considered the best available technology and has been demonstrated in prototype at SEG. The resultant metal for resale would have contamination levels between 0.3 and 20 becquerel per gram (Bq/g). The health hazards associated with release of the decontaminated nickel are minimal. The activity concentration of the end product would be further reduced when the nickel is combined with other metals to make stainless steel. Low-level radioactive waste from the SEG decontamination process, estimated to be approximately 382 m 3 (12,730 ft), would be shipped to a licensed commercial or DOE disposal facility. If the waste were packaged in 0.23 m 3 -(7.5 ft 3 -) capacity drums, approximately 1,500 to 1,900 drums would be transported over the 48-month contract period. Impacts from the construction of decontamination facilities and the selected site are minimal

  14. Nickel-based anodic electrocatalysts for fuel cells and water splitting

    Science.gov (United States)

    Chen, Dayi

    Our world is facing an energy crisis, so people are trying to harvest and utilize energy more efficiently. One of the promising ways to harvest energy is via solar water splitting to convert solar energy to chemical energy stored in hydrogen. Another of the options to utilize energy more efficiently is to use fuel cells as power sources instead of combustion engines. Catalysts are needed to reduce the energy barriers of the reactions happening at the electrode surfaces of the water-splitting cells and fuel cells. Nickel-based catalysts happen to be important nonprecious electrocatalysts for both of the anodic reactions in alkaline media. In alcohol fuel cells, nickel-based catalysts catalyze alcohol oxidation. In water splitting cells, they catalyze water oxidation, i.e., oxygen evolution. The two reactions occur in a similar potential range when catalyzed by nickel-based catalysts. Higher output current density, lower oxidation potential, and complete substrate oxidation are preferred for the anode in the applications. In this dissertation, the catalytic properties of nickel-based electrocatalysts in alkaline medium for fuel oxidation and oxygen evolution are explored. By changing the nickel precursor solubility, nickel complex nanoparticles with tunable sizes on electrode surfaces were synthesized. Higher methanol oxidation current density is achieved with smaller nickel complex nanoparticles. DNA aggregates were used as a polymer scaffold to load nickel ion centers and thus can oxidize methanol completely at a potential about 0.1 V lower than simple nickel electrodes, and the methanol oxidation pathway is changed. Nickel-based catalysts also have electrocatalytic activity towards a wide range of substrates. Experiments show that methanol, ethanol, glycerol and glucose can be deeply oxidized and carbon-carbon bonds can be broken during the oxidation. However, when comparing methanol oxidation reaction to oxygen evolution reaction catalyzed by current nickel

  15. Directional Darwinian Selection in proteins.

    Science.gov (United States)

    McClellan, David A

    2013-01-01

    Molecular evolution is a very active field of research, with several complementary approaches, including dN/dS, HON90, MM01, and others. Each has documented strengths and weaknesses, and no one approach provides a clear picture of how natural selection works at the molecular level. The purpose of this work is to present a simple new method that uses quantitative amino acid properties to identify and characterize directional selection in proteins. Inferred amino acid replacements are viewed through the prism of a single physicochemical property to determine the amount and direction of change caused by each replacement. This allows the calculation of the probability that the mean change in the single property associated with the amino acid replacements is equal to zero (H0: μ = 0; i.e., no net change) using a simple two-tailed t-test. Example data from calanoid and cyclopoid copepod cytochrome oxidase subunit I sequence pairs are presented to demonstrate how directional selection may be linked to major shifts in adaptive zones, and that convergent evolution at the whole organism level may be the result of convergent protein adaptations. Rather than replace previous methods, this new method further complements existing methods to provide a holistic glimpse of how natural selection shapes protein structure and function over evolutionary time.

  16. Analysis of drug-protein binding using on-line immunoextraction and high-performance affinity microcolumns: Studies with normal and glycated human serum albumin.

    Science.gov (United States)

    Matsuda, Ryan; Jobe, Donald; Beyersdorf, Jared; Hage, David S

    2015-10-16

    A method combining on-line immunoextraction microcolumns with high-performance affinity chromatography (HPAC) was developed and tested for use in examining drug-protein interactions with normal or modified proteins. Normal human serum albumin (HSA) and glycated HSA were used as model proteins for this work. High-performance immunoextraction microcolumns with sizes of 1.0-2.0 cm × 2.1mm i.d. and containing anti-HSA polyclonal antibodies were developed and tested for their ability to bind normal HSA or glycated HSA. These microcolumns were able to extract up to 82-93% for either type of protein at 0.05-0.10 mL/min and had a binding capacity of 0.34-0.42 nmol HSA for a 1.0 cm × 2.1mm i.d. microcolumn. The immunoextraction microcolumns and their adsorbed proteins were tested for use in various approaches for drug binding studies. Frontal analysis was used with the adsorbed HSA/glycated HSA to measure the overall affinities of these proteins for the drugs warfarin and gliclazide, giving comparable values to those obtained previously using similar protein preparations that had been covalently immobilized within HPAC columns. Zonal elution competition studies with gliclazide were next performed to examine the specific interactions of this drug at Sudlow sites I and II of the adsorbed proteins. These results were also comparable to those noted in prior work with covalently immobilized samples of normal HSA or glycated HSA. These experiments indicated that drug-protein binding studies can be carried out by using on-line immunoextraction microcolumns with HPAC. The same method could be used in the future with clinical samples and other drugs or proteins of interest in pharmaceutical studies or biomedical research. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Nickel elution properties of contemporary interatrial shunt closure devices.

    Science.gov (United States)

    Verma, Divya Ratan; Khan, Muhammad F; Tandar, Anwar; Rajasekaran, Namakkal S; Neuharth, Renée; Patel, Amit N; Muhlestein, Joseph B; Badger, Rodney S

    2015-02-01

    We sought to compare nickel elution properties of contemporary interatrial shunt closure devices in vitro. There are two United States Food and Drug Administration (FDA)-approved devices for percutaneous closure of secundum atrial septal defect: the Amplatzer septal occluder (ASO; St Jude Medical Corporation) and Gore Helex septal occluder (HSO; W.L. Gore & Associates). The new Gore septal occluder (GSO) device is in clinical trials. These are also used off-label for patent foramen ovale closure in highly selected patients. These devices have high nickel content. Nickel allergy is the most common reason for surgical device explantation. Nickel elution properties of contemporary devices remain unknown. We compared nickel elution properties of 4 devices - ASO, GSO, HSO, and sternal wire (SW) - while Dulbecco's phosphate-buffered saline (DPBS) served as control. Three samples of each device were submerged in DPBS. Nickel content was measured at 14 intervals over 90 days. Nickel elution at 24 hours, compared to control (0.005 ± 0.0 mg/L), was significantly higher for ASO (2.98 ± 1.65 mg/L; P=.04) and SW (0.03 ± 0.014 mg/L; P=.03). Nickel levels at 90 days, compared to control (0.005 ± 0.0 mg/L) and adjusting for multiple comparisons, were significantly higher for ASO (19.80 ± 2.30 mg/L; P=.01) and similar for HSO (P=.34), GSO (P=.34), and SW (P=.34). ASO had significantly higher nickel elution compared to HSO, GSO, and SW (P=.01). There is substantial variability in nickel elution; devices with less exposed nickel (HSO and GSO) have minimal elution. The safety of low nickel elution devices in patients with nickel allergy needs to be evaluated in prospective trials.

  18. Multilayer affinity adsorption of albumin on polymer brushes modified membranes in a continuous-flow system.

    Science.gov (United States)

    Hu, Meng-Xin; Li, Xiang; Li, Ji-Nian; Huang, Jing-Jing; Ren, Ge-Rui

    2018-02-23

    Polymer brushes modified surfaces have been widely used for protein immobilization and isolation. Modification of membranes with polymer brushes increases the surface concentration of affinity ligands used for protein binding. Albumin is one of the transporting proteins and shows a high affinity to bile acids. In this work, the modified membranes with cholic acid-containing polymer brushes can be facilely prepared by the immobilization of cholic acid on the poly(2-hydroxyethyl methacrylate) grafted microporous polypropylene membranes (MPPMs) for affinity adsorption of albumin. ATR/FT-IR and X-ray photoelectron spectroscopy were used to characterize the chemical composition of the modified membranes. Water contact angle measurements were used to analyze the hydrophilic/hydrophobic properties of the membrane surface. The modified MPPMs show a high affinity to albumin and have little non-specific adsorption of hemoglobin. The dynamic binding capacity of albumin in the continous-flow system increases with the cycle number and feed rate as the binding degree of cholic acid is moderate. The highest binding capacity of affinity membranes is about 52.49 g/m 2 membrane, which is about 24 times more than the monolayer binding capacity. These results reveal proteins could be captured in multilayers by the polymer brushes containing affinity ligands similar to the polymer brushes containing ion-exchange groups, which open up the potential of the polymer brushes containing affinity ligands in protein or another components separation. And the cholic acid containing polymer brushes modified membranes has the promising potential for albumin separation and purification rapidly from serum or fermented solution in medical diagnosis and bioseparation. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Selecting a Control Strategy for Plug and Process Loads

    Energy Technology Data Exchange (ETDEWEB)

    Lobato, C.; Sheppy, M.; Brackney, L.; Pless, S.; Torcellini, P.

    2012-09-01

    Plug and Process Loads (PPLs) are building loads that are not related to general lighting, heating, ventilation, cooling, and water heating, and typically do not provide comfort to the building occupants. PPLs in commercial buildings account for almost 5% of U.S. primary energy consumption. On an individual building level, they account for approximately 25% of the total electrical load in a minimally code-compliant commercial building, and can exceed 50% in an ultra-high efficiency building such as the National Renewable Energy Laboratory's (NREL) Research Support Facility (RSF) (Lobato et al. 2010). Minimizing these loads is a primary challenge in the design and operation of an energy-efficient building. A complex array of technologies that measure and manage PPLs has emerged in the marketplace. Some fall short of manufacturer performance claims, however. NREL has been actively engaged in developing an evaluation and selection process for PPLs control, and is using this process to evaluate a range of technologies for active PPLs management that will cap RSF plug loads. Using a control strategy to match plug load use to users' required job functions is a huge untapped potential for energy savings.

  20. Investigation on biomolecular interactions of nickel(II) complexes with monoanionic bidentate ligands

    Science.gov (United States)

    Jayamani, Arumugam; Sethupathi, Murugan; Ojwach, Stephen O.; Sengottuvelan, Nallathambi

    2018-01-01

    Reactions of monoanionic bidentate ligands 5-methylsalicylaldehyde (5-msal), 5-bromosalicylaldehyde (5-brsal), 5-nitrosalicylaldehyde (5-nsal) and 2-hydroxy-1-naphthaldehyde (2-hnap) with nickel perchlorate hexahydrate produced nickel(II) complexes 1-4, respectively. Single crystal X-ray analyses of complexes 1 and 2 confirmed bidentate mode of the ligands with O˄O coordination to give square planar geometry around nickel atoms. Complexes 1-4 showed one quasi-reversible redox peak at cathodic region (-0.67 to -0.80 V) and one redox peak at anodic region (+1.08 to +1.44 V) assignable to the Ni(II)/Ni(I) and Ni(II)/Ni(III) redox couples, respectively. The complexes exhibited good bovine serum albumin (BSA) binding abilities with a maximum binding constant of 1.96 × 105 M-1. The binding of complexes with calf thymus DNA (ctDNA) showed that the binding affinity is consistent with an increase in steric bulk of the ligands. The nuclease activity of the complexes showed efficient oxidative cleavage in the presence of hydrogen peroxide as an oxidizing agent. The complexes showed higher zone of inhibition when screened for antimicrobial activity against bacteria and human pathogenic fungi.

  1. NICKEL PLATING PROCESS

    Science.gov (United States)

    Hoover, T.B.; Zava, T.E.

    1959-05-12

    A simplified process is presented for plating nickel by the vapor decomposition of nickel carbonyl. In a preferred form of the invention a solid surface is nickel plated by subjecting the surface to contact with a mixture containing by volume approximately 20% nickel carbonyl vapor, 2% hydrogen sulfide and .l% water vapor or 1% oxygen and the remainder carbon dioxide at room temperature until the desired thickness of nickel is obtained. The advantage of this composition over others is that the normally explosive nickel carbonyl is greatly stabilized.

  2. Template-Assisted Synthesis and Characterization of Passivated Nickel Nanoparticles

    Directory of Open Access Journals (Sweden)

    Al-Omari IA

    2010-01-01

    Full Text Available Abstract Potential applications of nickel nanoparticles demand the synthesis of self-protected nickel nanoparticles by different synthesis techniques. A novel and simple technique for the synthesis of self-protected nickel nanoparticles is realized by the inter-matrix synthesis of nickel nanoparticles by cation exchange reduction in two types of resins. Two different polymer templates namely strongly acidic cation exchange resins and weakly acidic cation exchange resins provided with cation exchange sites which can anchor metal cations by the ion exchange process are used. The nickel ions which are held at the cation exchange sites by ion fixation can be subsequently reduced to metal nanoparticles by using sodium borohydride as the reducing agent. The composites are cycled repeating the loading reduction cycle involved in the synthesis procedure. X-Ray Diffraction, Scanning Electron Microscopy, Transmission Electron microscopy, Energy Dispersive Spectrum, and Inductively Coupled Plasma Analysis are effectively utilized to investigate the different structural characteristics of the nanocomposites. The hysteresis loop parameters namely saturation magnetization and coercivity are measured using Vibrating Sample Magnetometer. The thermomagnetization study is also conducted to evaluate the Curie temperature values of the composites. The effect of cycling on the structural and magnetic characteristics of the two composites are dealt in detail. A comparison between the different characteristics of the two nanocomposites is also provided.

  3. The influence of nickel sulphate on some physiological aspects of two cultivars of Raphanus sativus L.

    Directory of Open Access Journals (Sweden)

    Latif Helmy H.

    2010-01-01

    Full Text Available In this study two cultivars of radish Raphanus sativus cv. longipinnatus (white radish and Raphanus sativus cv. Cherry Belle (red radish were treated with different concentrations of nickel sulphate (0.0-50-100-150-200 ppm. The fresh and dry weight of shoots and roots, photosynthetic pigments, some antioxidant enzymes, total carbohydrates, total proteins and the SDS-PAGE protein profile of both cultivars were determined after 32 days. The results showed that increasing nickel sulphate concentrations decreased the fresh and dry weights of the shoots and roots, photosynthetic pigments, total carbohydrates and total protein in both cultivars. Higher concentrations of nickel sulphate increased the activity of catalase, peroxidase and polypenol oxidase. Electrophoresis banding profiles of proteins revealed qualitative and quantitative changes, and also the appearance or disappearance of some bands of the two cultivars. .

  4. Attentional selection within and across hemispheres: implications for the perceptual load theory.

    Science.gov (United States)

    Wei, Ping; Kang, Guanlan; Zhou, Xiaolin

    2013-03-01

    The perceptual load of a given task affects attentional selection, with the selection occurring earlier when the load is high and later when the load is low. Recent evidence suggests that local competitive interaction may underlie the perceptual load effect and determine to what extent a task-irrelevant distractor is processed. Here, we asked participants to search for a target bar among homogeneously oriented bars (the low load conditions) or heterogeneously oriented bars (the high load conditions) in the central display, while ignoring a congruent or incongruent flanker bar presented to the left or right side of the central display, or a bar presented at one of the six positions outer to the central display. Importantly, we differentiated conditions in which the target in the central display and the peripheral flanker was presented within the same hemifield or across different hemifields. Results showed a significant flanker effect for the low load condition, but not for the high load condition, when the target and the flanker were across hemifields. However, when the target and the flanker were presented within the same hemifield, there was no flanker effect for either low or high load conditions. These findings demonstrate that the ability to ignore the task-irrelevant distractor is affected by local competition within hemisphere and that the perceptual load theory needs to be supplemented with detailed analysis of cognitive processes and mechanisms underlying the consumption of attentional resources.

  5. High affinity radiopharmaceuticals based upon lansoprazole for PET imaging of aggregated tau in Alzheimer's disease and progressive supranuclear palsy: synthesis, preclinical evaluation, and lead selection.

    Science.gov (United States)

    Fawaz, Maria V; Brooks, Allen F; Rodnick, Melissa E; Carpenter, Garrett M; Shao, Xia; Desmond, Timothy J; Sherman, Phillip; Quesada, Carole A; Hockley, Brian G; Kilbourn, Michael R; Albin, Roger L; Frey, Kirk A; Scott, Peter J H

    2014-08-20

    Abnormally aggregated tau is the hallmark pathology of tauopathy neurodegenerative disorders and is a target for development of both diagnostic tools and therapeutic strategies across the tauopathy disease spectrum. Development of carbon-11- or fluorine-18-labeled radiotracers with appropriate affinity and specificity for tau would allow noninvasive quantification of tau burden using positron emission tomography (PET) imaging. We have synthesized [(18)F]lansoprazole, [(11)C]N-methyl lansoprazole, and [(18)F]N-methyl lansoprazole and identified them as high affinity radiotracers for tau with low to subnanomolar binding affinities. Herein, we report radiosyntheses and extensive preclinical evaluation with the aim of selecting a lead radiotracer for translation into human PET imaging trials. We demonstrate that [(18)F]N-methyl lansoprazole, on account of the favorable half-life of fluorine-18 and its rapid brain entry in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau over amyloid, is the lead compound for progression into clinical trials.

  6. Affine and quasi-affine frames for rational dilations

    DEFF Research Database (Denmark)

    Bownik, Marcin; Lemvig, Jakob

    2011-01-01

    In this paper we extend the investigation of quasi-affine systems, which were originally introduced by Ron and Shen [J. Funct. Anal. 148 (1997), 408-447] for integer, expansive dilations, to the class of rational, expansive dilations. We show that an affine system is a frame if, and only if......, the corresponding family of quasi-affine systems are frames with uniform frame bounds. We also prove a similar equivalence result between pairs of dual affine frames and dual quasi-affine frames. Finally, we uncover some fundamental differences between the integer and rational settings by exhibiting an example...

  7. Identification of Eupatilin from Artemisia argyi as a Selective PPARα Agonist Using Affinity Selection Ultrafiltration LC-MS

    Directory of Open Access Journals (Sweden)

    Yongsoo Choi

    2015-07-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs are key nuclear receptors and therapeutic targets for the treatment of metabolic diseases through the regulation of insulin resistance, diabetes, and dyslipidemia. Although a few drugs that target PPARs have been approved, more diverse and novel PPAR ligands are necessary to improve the safety and efficacy of available drugs. To expedite the search for new natural agonists of PPARs, we developed a screening assay based on ultrafiltration liquid chromatography-mass spectrometry (LC-MS that is compatible with complex samples such as dietary foods or botanical extracts. The known PPARα and/or PPARγ ligands resveratrol and rosiglitazone were used as positive controls to validate the developed method. When applied to the screening of an Artemisia argyi extract, eupatilin was identified as a selective PPARα ligand. A PPAR competitive binding assay based on FRET detection also confirmed eupatilin as a selective PPARα agonist exhibiting a binding affinity of 1.18 μM (IC50. Furthermore, eupatilin activation of the transcriptional activity of PPARα was confirmed using a cell-based transactivation assay. Thus, ultrafiltration LC-MS is a suitable assay for the identification of PPAR ligands in complex matrixes such as extracts of dietary foods and botanicals.

  8. A control on hydrophobic and hydrophilic interactions between HEWL and metal Schiff-base complexes comprising of different metal ions and ligands

    Energy Technology Data Exchange (ETDEWEB)

    Koley Seth, Banabithi; Ray, Aurkie; Basu, Samita, E-mail: samita.basu@saha.ac.in

    2015-05-15

    The structural effects of different copper(II) and nickel(II) Schiff base complexes on hen egg white lysozyme (HEWL) have been investigated through steady state and time resolved absorption and fluorescence, and circular dichroism spectroscopy. The Schiff base ligands with N{sub 4} donor atoms show both hydrophobic and hydrophilic interactions, however hydrophilic interaction prevails with ligands having N{sub 2}O{sub 2} donor atoms. Variation of metal ions from Cu{sup 2+} to Ni{sup 2+} with each type of Schiff base ligand increases the probability of hydrophilic over hydrophobic interactions, which supports their significance in regulating the binding affinity between HEWL and metal complexes. On photo-excitation the complexes comprising of Cu{sup 2+} ion instead of Ni{sup 2+} ion and ligands with N{sub 4} donor system rather than N{sub 2}O{sub 2} donor system, increases the probability of intersystem crossing to populate the corresponding triplet state as observed from laser flash photolysis study. The better binding affinity of nickel complexes with different selectivities compared to copper complexes towards HEWL emphasizes the potentiality of less explored nickel complexes in drug–protein interactions. - Highlights: • Ni{sup II} and Cu{sup II} -Schiff base complexes bind hen egg white lysozyme spontaneously. • Both hydrophobic and hydrophilic interactions are effective for N{sub 4} ligands. • For N{sub 2}O{sub 2} ligands the hydrophilic is predominant over hydrophobic interaction. • Binding affinity and selectivity of Ni{sup II}-complexes are better than Cu{sup II}-complexes. • Replacement of Cu{sup 2+} by Ni{sup 2+} in a ligand enhances chance of hydrophilic interaction.

  9. Polyamide 6/nickel ferrite composites: morphological and structural evaluation; Compositos poliamida 6 /ferrita de niquel: avaliacao morfologica e estrutural

    Energy Technology Data Exchange (ETDEWEB)

    Menezes, P.C.F.; Santos, P.T.A.; Wanderley, A.S.D.; Costa, A.C.F.M.; Araujo, E.M., E-mail: patricia.fernandes24@hotmail.com [Universidade Federal de Campina Grande (UFCG), Campina Grande, PB (Brazil). Unidade Academica de Engenharia de Materiais

    2011-07-01

    This study aims to evaluate the influence of particle size of the load on the structure and morphology of polyamide composite 6/ferrita nickel. To obtain the composite, the polyamide 6 was previously vacuum dried at 80 ° C/48h to eliminate moisture, and then the load was incorporated as powders and as synthesized after calcination at 900°C in the mass concentration of 50%. Subsequently, the mixture was compressed using three tons to obtain the composites, which were characterized by XRD and SEM. XRD results showed the characteristic peaks of polyamide 6 and ferrite. An increase in crystallinity was observed for the composite loaded calcined at 900° C. SEM results showed a good dispersion of nickel ferrite loading in the matrix, the presence of larger clusters and evenly distributed load for the composite calcined at 900 ° C. (author)

  10. Carbon deposition on nickel ferrites and nickel-magnetite surfaces

    International Nuclear Information System (INIS)

    Allen, G.C.; Jutson, J.A.

    1988-06-01

    Carbon deposition on Commercial Advanced Gas-Cooled Reactor (CAGR) fuel cladding and heat exchanger surfaces lowers heat transfer efficiency and increases fuel pin temperatures. Several types of deposit have been identified including both thin dense layers and also low density columnar deposits with filamentary or convoluted laminar structure. The low-density types are often associated with particles containing iron, nickel or manganese. To identify the role of nickel in the deposition process surfaces composed of nickel-iron spinels or metallic nickel/magnetite mixtures have been exposed to γ radiation in a gas environment simulating that in the reactor. Examination of these surfaces by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) have shown that while metallic nickel (Ni(O)) catalyses the formation of filamentary low density carbon deposits, the presence of divalent nickel (Ni(II)) sites in spinel type oxides is associated only with dense deposits. (author)

  11. Nb effect in the nickel oxide-catalyzed low-temperature oxidative dehydrogenation of ethane

    KAUST Repository

    Zhu, Haibo

    2012-01-01

    A method for the preparation of NiO and Nb-NiO nanocomposites is developed, based on the slow oxidation of a nickel-rich Nb-Ni gel obtained in citric acid. The resulting materials have higher surface areas than those obtained by the classical evaporation method from nickel nitrate and ammonium niobium oxalate. These consist in NiO nanocrystallites (7-13 nm) associated, at Nb contents >3 at.%., with an amorphous thin layer (1-2 nm) of a niobium-rich mixed oxide with a structure similar to that of NiNb 2O 6. Unlike bulk nickel oxides, the activity of these nanooxides for low-temperature ethane oxidative dehydrogenation (ODH) has been related to their redox properties. In addition to limiting the size of NiO crystallites, the presence of the Nb-rich phase also inhibits NiO reducibility. At Nb content >5 at.%, Nb-NiO composites are thus less active for ethane ODH but more selective, indicating that the Nb-rich phase probably covers part of the unselective, non-stoichiometric, active oxygen species of NiO. This geometric effect is supported by high-resolution transmission electron microscopy observations. The close interaction between NiO and the thin Nb-rich mixed oxide layer, combined with possible restructuration of the nanocomposite under ODH conditions, leads to significant catalyst deactivation at high Nb loadings. Hence, the most efficient ODH catalysts obtained by this method are those containing 3-4 at.% Nb, which combine high activity, selectivity, and stability. The impact of the preparation method on the structural and catalytic properties of Nb-NiO nanocomposites suggests that further improvement in NiO-catalyzed ethane ODH can be expected upon optimization of the catalyst. © 2011 Elsevier Inc. All rights reserved.

  12. Ni nanoparticles decorated onto graphene oxide with SiO2 as interlayer for high performance on histidine-rich protein separation

    Science.gov (United States)

    Yang, Xiaodan; Zhang, Min; Zheng, Jing; Li, Weizhen; Gan, Wenjun; Xu, Jingli; Hayat, Tasawar; Alharbi, Njud S.; Yang, Fan

    2018-05-01

    Sandwich-like structure of graphene oxide (GO) @SiO2@C-Ni nanosheets were prepared by combining an extended stöber method with subsequent carbonization treatment, in which polydopamine was used as reducing agent and carbon source. Firstly, the GO nanosheets were covered with SiO2 interlayer and finally coated with a outer shell of nickel ion doped polydopamine (PDA-Ni2+) with an extended stöber method. Followed by a carbonization to produce the GO@SiO2@C-Ni sheets with metallic nickel nanoparticles embedded in PDA-derived thin graphic carbon layer. Notably, silica interlayer played a vital role in the formation of such GO@SiO2@C-Ni sheets. Without the protection of SiO2, the hydrophobic graphene@C-Ni composites were obtained instead. While with silica layer as the spacer, the obtained hydrophilic GO@SiO2@C-Ni composites were not only well dispersed in the solution, but also can be adjusted in terms of the size and density of Ni nanoparticles (NPs) on surface by changing the calcination temperature or the molar ratio between dopamine and nickel salt. Furthermore, nickel nanoparticles decorated on GO@SiO2 sheets were employed to enrich His-rich proteins (BHb and BSA) via specific metal affinity force between polyhistidine groups and nickel nanoparticles.

  13. Phase Transformations in Nickel base Superalloy Inconel 718 during Cyclic Loading at High Temperature

    Directory of Open Access Journals (Sweden)

    Michal Jambor

    2017-06-01

    Full Text Available Nickel base superalloys are hi-tech materials intended for high temperature applications. This property owns a complex microstructure formed by matrix of Ni and variety of precipitates. The type, form and the amount of these phases significantly affect the resulting properties of these alloys. At sufficiently long exposure to high temperatures, the transformation phase can occur, which can lead to degradation of properties of these alloys. A cyclic plastic deformation can accelerate these changes, and they could occur at significantly lower temperatures or in shorter time of exposure. The aim of this study is to describe phase transformation, which can occur by a cyclic plastic deformation at high temperatures in nickel base superalloy Inconel 718.

  14. Nickel-hydrogen battery; Nikkeru/suiso batteri

    Energy Technology Data Exchange (ETDEWEB)

    Kuwajima, S. [National Space Development Agency, Tokyo (Japan)

    1996-07-01

    In artificial satellites, electric power is supplied from batteries loaded on them, when sun light can not be rayed on the event of equinoxes. Thus, research and development was started as early as 1970s for light and long-life batteries. Nickel-hydrogen batteries have been used on practical satellites since middle of 1980s. Whereas the cathode reaction of this battery is the same as that of a conventional nickel-cadmium battery, the anode reaction is different in that it involves decomposition and formation of water, generating hydrogen and consuming it. Hydrogen is stored in a state of pressurized gas within the battery vessel. The shape of this vessel is of a bomb, whose size for the one with capacity of 35 Ah is 8cm in diameter and 18cm in length. On a satellite, this one is assembled into a set of 16 ones. National Space Development Agency of Japan has been conducting the evaluation test for nickel-hydrogen batteries in a long term range. It was made clear that the life-determinant factor is related to the inner electrode, not to the vessel. Performance data on long-term endurance of materials to be used have been accumulated also in the agency. 2 figs.

  15. Preparation of GST Fusion Proteins.

    Science.gov (United States)

    Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

    2007-04-01

    INTRODUCTIONThis protocol describes the preparation of glutathione-S-transferase (GST) fusion proteins, which have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis.

  16. Molecular basis of a high affinity murine interleukin-5 receptor.

    OpenAIRE

    Devos, R; Plaetinck, G; Van der Heyden, J; Cornelis, S; Vandekerckhove, J; Fiers, W; Tavernier, J

    1991-01-01

    The mouse interleukin-5 receptor (mIL-5R) consists of two components one of which, the mIL-5R alpha-chain, binds mIL-5 with low affinity. Recently we demonstrated that monoclonal antibodies (Mabs) recognizing the second mIL-5R beta-chain, immunoprecipitate a p130-140 protein doublet which corresponds to the mIL-3R and the mIL-3R-like protein, the latter chain for which so far no ligand has been identified. In this study we show that a high affinity mIL-5R can be reconstituted on COS1 cells by...

  17. Nickel compounds induce histone ubiquitination by inhibiting histone deubiquitinating enzyme activity

    International Nuclear Information System (INIS)

    Ke Qingdong; Ellen, Thomas P.; Costa, Max

    2008-01-01

    Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis

  18. Dual Mode Fluorophore-Doped Nickel Nitrilotriacetic Acid-Modified Silica Nanoparticles Combine Histidine-Tagged Protein Purification with Site-Specific Fluorophore Labeling

    OpenAIRE

    Kim, Sung Hoon; Jeyakumar, M.; Katzenellenbogen, John A.

    2007-01-01

    We present the first example of a fluorophore-doped nickel chelate surface- modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethylrhodamine (TMR)-doped silica nanoparticles, estimated to contain 700–900 TMRs per ca. 23-nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni+2. Silica-embedded TMR retains very high quantum yield, is resistant to quenc...

  19. The CRAPome: a contaminant repository for affinity purification–mass spectrometry data

    NARCIS (Netherlands)

    Mellacheruvu, D.; Wright, Z.; Couzens, A.L.; Low, T.Y.|info:eu-repo/dai/nl/411298437; Halim, V.A.|info:eu-repo/dai/nl/326157441; Heck, A.J.R.|info:eu-repo/dai/nl/105189332; Mohammed, S.|info:eu-repo/dai/nl/30483632X; Nesvizhskii, A.

    2013-01-01

    Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background

  20. Removal of lead, mercury and nickel using the yeast Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Cherlys Infante J.

    2014-06-01

    Full Text Available Objective. In this study the biomass of the yeast Saccharomyces cerevisiae was used to remove lead, mercury and nickel in the form of ions dissolved in water. Materials and methods. Synthetic solutions were prepared containing the three heavy metals, which were put in contact with viable microorganisms at different conditions of pH, temperature, aeration and agitation. Results. Both individual variables and the interaction effects influenced the biosorption process. Throughout the experimental framework it was observed that the biomass of Saccharomyces cerevisiae removed a higher percentage of lead (86.4% as compared to mercury and nickel (69.7 and 47.8% respectively. When the pH was set at a value of 5 the effect was positive for all three metals. Conclusions. pH was the variable that had a greater influence on the biosorption of lead on the biomass of Saccharomyces cerevisiae. The affinity of the heavy metals for the biomass followed the order Pb>Hg>Ni.

  1. The EU Nickel Directive revisited--future steps towards better protection against nickel allergy

    DEFF Research Database (Denmark)

    Thyssen, Jacob P; Uter, Wolfgang; McFadden, John

    2011-01-01

    In July 2001, the EU Nickel Directive came into full force to protect European citizens against nickel allergy and dermatitis. Prior to this intervention, Northern European governments had already begun to regulate consumer nickel exposure. According to part 2 of the EU Nickel Directive and the D......In July 2001, the EU Nickel Directive came into full force to protect European citizens against nickel allergy and dermatitis. Prior to this intervention, Northern European governments had already begun to regulate consumer nickel exposure. According to part 2 of the EU Nickel Directive...... and the Danish nickel regulation, consumer items intended to be in direct and prolonged contact with the skin were not allowed to release more than 0.5 µg nickel/cm2/week. It was considered unlikely that nickel allergy would disappear altogether as a proportion of individuals reacted below the level defined...

  2. Protective effects of zinc acetate toward the toxicity of nickelous acetate in rats

    International Nuclear Information System (INIS)

    Waalkes, M.P.; Kasprzak, K.S.; Ohshima, M.; Poirier, L.A.

    1985-01-01

    This study was designed to determine the effects of zinc pretreatment on the acute toxicity of nickel. Male Fischer rats received either nickel alone (i.p.), zinc alone (s.c.), zinc plus nickel, or saline (i.p. and s.c.; controls). Zinc pretreatment significantly increased the 14-day survival of nickel-related rats. Zinc did not, however, prevent the reduction in weight gain over 2 weeks seen with nickel treatment. Histopathologically, at 120 h following nickel exposure, kidneys in the group receiving nickel alone generally showed moderate nephropathy (multifocal proximal tubule degeneration with necrosis) while in the zinc plus nickel group the nephropathy was generally mild. Zinc pretreatment had no apparent effect on the pharmacokinetics of nickel over 24 h as assessed by urinary excretion, blood levels or organ distribution. Zinc pretreatment also did not alter the subcellular distribution of renal nickel 6 h after nickel exposure. Enhanced synthesis of metallothionein did not appear to play a critical role in the reduction of nickel toxicity, since renal concentrations of this metalbuilding protein, although elevated compared to control, were not different in rats receiving zinc and nickel or zinc alone. Zinc pretreatment did, however, have marked effect on nickel-induced hyperglycemia, reducing both the duration and severity of elevated blood glucose levels. Results of the study show that zinc can prevent some of the toxic effects of nickel and that the mechanism of this action does not appear to involve either metalothionein or alterations in the pharmacokinetics of nickel. (author)

  3. De novo biofilm community assembly from tap water source communities favors Nitrotoga over Nitrospira under elevated nitrite surface loading

    DEFF Research Database (Denmark)

    Kinnunen, Marta; Dechesne, Arnaud; Albrechtsen, Hans-Jørgen

    -through biofilm system to continuous immigration from a tap water metacommunity while applying different nitrite surface loading rates. After 63 days of operation, we extracted biofilms and analyzed the community composition via Illumina MiSeq targeting the 16S rRNA gene. Previous studies have shown...... that Nitrospira is the dominant nitrite oxidizing genus in low nitrite environments. Hence, we postulated that by elevating the nitrite surface loading we would select for NOB with lower nitrite affinity than Nitrospira. We observed different dominant NOB species under different loading rates. While...... in the metacommunity, Nitrotoga and Nitrospira were found at near equal abundances, in the biofilm community, elevated nitrite loading strongly selected for Nitrotoga over Nitrospira. The biofilms were also significantly different in their alpha-diversity (pdiversity, and the evenness and richness...

  4. Electrolytic Recovery of Nickel from Spent Electroless Nickel Bath Solution

    Directory of Open Access Journals (Sweden)

    R. Idhayachander

    2010-01-01

    Full Text Available Plating industry is one of the largest polluting small scale industries and nickel plating is among the important surface finishing process in this industry. The waste generated during this operation contains toxic nickel. Nickel removal and recovery is of great interest from spent bath for environmental and economic reasons. Spent electroless nickel solution from a reed relay switch manufacturing industry situated in Chennai was taken for electrolytic recovery of nickel. Electrolytic experiment was carried out with mild steel and gold coated mild steel as cathode and the different parameters such as current density, time, mixing and pH of the solution were varied and recovery and current efficiency was studied. It was noticed that there was an increase in current efficiency up to 5 A/dm2 and after that it declines. There is no significant improvement with mixing but with modified cathode there was some improvement. Removal of nickel from the spent electroless nickel bath was 81.81% at 5 A/dm2 and pH 4.23. Under this condition, the content of nickel was reduced to 0.94 g/L from 5.16 g/L. with 62.97% current efficiency.

  5. Development of nickel membranes deposited on ceramic materials by electroless plating: studies of the hydrogen perm-selectivity properties at elevated temperatures

    International Nuclear Information System (INIS)

    Amer, J.

    2008-09-01

    The main objective of this work was to synthesize nickel based membranes by electroless plating on materials such as alumina-α, alumina-γ and zirconia with various textures and to determine their hydrogen perm-selectivity at high temperatures. The synthesis of metal films of high purity (≥ 99% mass Ni) resulting from the choice of hydrazine with its dual role of reducing and complexing agent has revealed that the diameter of pores on the surface support has an impact on the quality of metal adherence. The various contributions of hydrogen transport through these composite membranes at low temperatures (Knudsen and surface diffusion) and at high temperatures (Knudsen and activated diffusion) was established. At its implementation in a membrane reactor (reaction of propane dehydrogenation), the layer of nickel showed a very good resistance to coking. (author)

  6. Human exposure to nickel

    Energy Technology Data Exchange (ETDEWEB)

    Grandjean, P

    1984-01-01

    In order of abundance in the earth's crust, nickel ranks as the 24th element and has been detected in different media in all parts of the biosphere. Thus, humans are constantly exposed to this ubiquitous element, though in variable amounts. Occupational exposures may lead to the retention of 100 micrograms of nickel per day. Environmental nickel levels depend particularly on natural sources, pollution from nickel-manufacturing industries and airborne particles from combustion of fossil fuels. Absorption from atmospheric nickel pollution is of minor concern. Vegetables usually contain more nickel than do other food items. Certain products, such as baking powder and cocoa powder, have been found to contain excessive amounts of nickel, perhaps related to nickel leaching during the manufacturing process. Soft drinking-water and acid beverages may dissolve nickel from pipes and containers. Scattered studies indicate a highly variable dietary intake of nickel, but most averages are about 200-300 micrograms/day. In addition, skin contact to a multitude of metal objects may be of significance to the large number of individuals suffering from contact dermatitis and nickel allergy. Finally, nickel alloys are often used in nails and prostheses for orthopaedic surgery, and various sources may contaminate intravenous fluids. Thus, human nickel exposure originates from a variety of sources and is highly variable. Occupational nickel exposure is of major significance, and leaching of nickel may add to dietary intakes and to cutaneous exposures. 79 references.

  7. Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites.

    OpenAIRE

    Erspamer, V; Melchiorri, P; Falconieri-Erspamer, G; Negri, L; Corsi, R; Severini, C; Barra, D; Simmaco, M; Kreil, G

    1989-01-01

    Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affi...

  8. Synthesis of Nickel and Nickel Hydroxide Nanopowders by Simplified Chemical Reduction

    Directory of Open Access Journals (Sweden)

    Jeerapan Tientong

    2014-01-01

    Full Text Available Nickel nanopowders were synthesized by a chemical reduction of nickel ions with hydrazine hydrate at pH ~12.5. Sonication of the solutions created a temperature of 54–65°C to activate the reduction reaction of nickel nanoparticles. The solution pH affected the composition of the resulting nanoparticles. Nickel hydroxide nanoparticles were formed from an alkaline solution (pH~10 of nickel-hydrazine complexed by dropwise titration. X-ray diffraction of the powder and the analysis of the resulting Williamson-Hall plots revealed that the particle size of the powders ranged from 12 to 14 nm. Addition of polyvinylpyrrolidone into the synthesis decreased the nickel nanoparticle size to approximately 7 nm. Dynamic light scattering and scanning electron microscopy confirmed that the particles were in the nanometer range. The structure of the synthesized nickel and nickel hydroxide nanoparticles was identified by X-ray diffraction and Fourier transform infrared spectroscopy.

  9. Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

    Science.gov (United States)

    Kuipers, Marjorie A.; Stasevich, Timothy J.; Sasaki, Takayo; Wilson, Korey A.; Hazelwood, Kristin L.; McNally, James G.; Davidson, Michael W.

    2011-01-01

    The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles. PMID:21220507

  10. Charge retention test experiences on Hubble Space Telescope nickel-hydrogen battery cells

    Science.gov (United States)

    Nawrocki, Dave E.; Driscoll, J. R.; Armantrout, J. D.; Baker, R. C.; Wajsgras, H.

    1993-01-01

    The Hubble Space Telescope (HST) nickel-hydrogen battery module was designed by Lockheed Missile & Space Co (LMSC) and manufactured by Eagle-Picher Ind. (EPI) for the Marshall Space Flight Center (MSFC) as an Orbital Replacement Unit (ORU) for the nickel-cadmium batteries originally selected for this low earth orbit mission. The design features of the HST nickel hydrogen battery are described and the results of an extended charge retention test are summarized.

  11. Towards Automated Binding Affinity Prediction Using an Iterative Linear Interaction Energy Approach

    Directory of Open Access Journals (Sweden)

    C. Ruben Vosmeer

    2014-01-01

    Full Text Available Binding affinity prediction of potential drugs to target and off-target proteins is an essential asset in drug development. These predictions require the calculation of binding free energies. In such calculations, it is a major challenge to properly account for both the dynamic nature of the protein and the possible variety of ligand-binding orientations, while keeping computational costs tractable. Recently, an iterative Linear Interaction Energy (LIE approach was introduced, in which results from multiple simulations of a protein-ligand complex are combined into a single binding free energy using a Boltzmann weighting-based scheme. This method was shown to reach experimental accuracy for flexible proteins while retaining the computational efficiency of the general LIE approach. Here, we show that the iterative LIE approach can be used to predict binding affinities in an automated way. A workflow was designed using preselected protein conformations, automated ligand docking and clustering, and a (semi-automated molecular dynamics simulation setup. We show that using this workflow, binding affinities of aryloxypropanolamines to the malleable Cytochrome P450 2D6 enzyme can be predicted without a priori knowledge of dominant protein-ligand conformations. In addition, we provide an outlook for an approach to assess the quality of the LIE predictions, based on simulation outcomes only.

  12. Grain-resolved analysis of localized deformation in nickel-titanium wire under tensile load.

    Science.gov (United States)

    Sedmák, P; Pilch, J; Heller, L; Kopeček, J; Wright, J; Sedlák, P; Frost, M; Šittner, P

    2016-08-05

    The stress-induced martensitic transformation in tensioned nickel-titanium shape-memory alloys proceeds by propagation of macroscopic fronts of localized deformation. We used three-dimensional synchrotron x-ray diffraction to image at micrometer-scale resolution the grain-resolved elastic strains and stresses in austenite around one such front in a prestrained nickel-titanium wire. We found that the local stresses in austenite grains are modified ahead of the nose cone-shaped buried interface where the martensitic transformation begins. Elevated shear stresses at the cone interface explain why the martensitic transformation proceeds in a localized manner. We established the crossover from stresses in individual grains to a continuum macroscopic internal stress field in the wire and rationalized the experimentally observed internal stress field and the topology of the macroscopic front by means of finite element simulations of the localized deformation. Copyright © 2016, American Association for the Advancement of Science.

  13. The EU Nickel Directive revisited--future steps towards better protection against nickel allergy

    DEFF Research Database (Denmark)

    Thyssen, Jacob P; Uter, Wolfgang; McFadden, John

    2011-01-01

    In July 2001, the EU Nickel Directive came into full force to protect European citizens against nickel allergy and dermatitis. Prior to this intervention, Northern European governments had already begun to regulate consumer nickel exposure. According to part 2 of the EU Nickel Directive...... by the EU Nickel Directive. Despite this, the EU Nickel Directive part 2 was expected to work as an operational limit that would sufficiently protect European consumers against nickel allergy and dermatitis. This review presents the accumulation of epidemiological studies that evaluated the possible effect...... and the Danish nickel regulation, consumer items intended to be in direct and prolonged contact with the skin were not allowed to release more than 0.5 µg nickel/cm2/week. It was considered unlikely that nickel allergy would disappear altogether as a proportion of individuals reacted below the level defined...

  14. The application of magnetic force differentiation for the measurement of the affinity of peptide libraries

    International Nuclear Information System (INIS)

    Shang Hao; Kirkham, Perry M.; Myers, Tina M.; Cassell, Gail H.; Lee, Gil U.

    2005-01-01

    A new method has been developed for measuring the binding affinity of phage displayed peptides and a target protein using magnetic particles. The specific interaction between the phage displayed peptides and the target protein was subject to a force generated by the magnetic particle. The binding affinity was obtained by analyzing the force-bond lifetime

  15. High Affinity Radiopharmaceuticals Based Upon Lansoprazole for PET Imaging of Aggregated Tau in Alzheimer’s Disease and Progressive Supranuclear Palsy: Synthesis, Preclinical Evaluation, and Lead Selection

    Science.gov (United States)

    2014-01-01

    Abnormally aggregated tau is the hallmark pathology of tauopathy neurodegenerative disorders and is a target for development of both diagnostic tools and therapeutic strategies across the tauopathy disease spectrum. Development of carbon-11- or fluorine-18-labeled radiotracers with appropriate affinity and specificity for tau would allow noninvasive quantification of tau burden using positron emission tomography (PET) imaging. We have synthesized [18F]lansoprazole, [11C]N-methyl lansoprazole, and [18F]N-methyl lansoprazole and identified them as high affinity radiotracers for tau with low to subnanomolar binding affinities. Herein, we report radiosyntheses and extensive preclinical evaluation with the aim of selecting a lead radiotracer for translation into human PET imaging trials. We demonstrate that [18F]N-methyl lansoprazole, on account of the favorable half-life of fluorine-18 and its rapid brain entry in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau over amyloid, is the lead compound for progression into clinical trials. PMID:24896980

  16. The importance of protein in leaf selection of folivorous primates.

    Science.gov (United States)

    Ganzhorn, Joerg U; Arrigo-Nelson, Summer J; Carrai, Valentina; Chalise, Mukesh K; Donati, Giuseppe; Droescher, Iris; Eppley, Timothy M; Irwin, Mitchell T; Koch, Flávia; Koenig, Andreas; Kowalewski, Martin M; Mowry, Christopher B; Patel, Erik R; Pichon, Claire; Ralison, Jose; Reisdorff, Christoph; Simmen, Bruno; Stalenberg, Eleanor; Starrs, Danswell; Terboven, Juana; Wright, Patricia C; Foley, William J

    2017-04-01

    Protein limitation has been considered a key factor in hypotheses on the evolution of life history and animal communities, suggesting that animals should prioritize protein in their food choice. This contrasts with the limited support that food selection studies have provided for such a priority in nonhuman primates, particularly for folivores. Here, we suggest that this discrepancy can be resolved if folivores only need to select for high protein leaves when average protein concentration in the habitat is low. To test the prediction, we applied meta-analyses to analyze published and unpublished results of food selection for protein and fiber concentrations from 24 studies (some with multiple species) of folivorous primates. To counter potential methodological flaws, we differentiated between methods analyzing total nitrogen and soluble protein concentrations. We used a meta-analysis to test for the effect of protein on food selection by primates and found a significant effect of soluble protein concentrations, but a non-significant effect for total nitrogen. Furthermore, selection for soluble protein was reinforced in forests where protein was less available. Selection for low fiber content was significant but unrelated to the fiber concentrations in representative leaf samples of a given forest. There was no relationship (either negative or positive) between the concentration of protein and fiber in the food or in representative samples of leaves. Overall our study suggests that protein selection is influenced by the protein availability in the environment, explaining the sometimes contradictory results in previous studies on protein selection. Am. J. Primatol. 79:e22550, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Selective recovery of Ag(I) coordination anion from simulate nickel electrolyte using corn stalk based adsorbent modified by ammonia–thiosemicarbazide

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Ying, E-mail: xiongying_1977@hotmail.com; Wan, Li; Xuan, Jing; Wang, Yongwei; Xing, Zhiqing; Shan, Weijun; Lou, Zhenning

    2016-01-15

    Highlights: • We developed a “green” corn stalk adsorbent treated by thiosemicarbazide–glutaraldehyde. • Adsorption mechanism of AgCl{sub i}{sup 1−i} could be electrostatic interaction with C−SH{sup +}−N and C=SH{sup +} group. • The gel could selectively adsorb AgCl{sub i}{sup 1−i} coordination anion from the Ag(I)–Cu(II)–Ni(II) simulate nickel electrolyte. - Abstract: In nickel electrolyte, Ag(I) was present at trace level concentration (10–20 mg L{sup −1}) and existed in the form of AgCl{sub i}{sup 1−i} coordination anion, instead of Ag{sup +} positive ion usually in several sources. In the present study, TSC-NH{sub 3}-OCS adsorbent based on natural corn stalk modified by ammonia (NH{sub 3})–thiosemicarbazide (TSC) was synthesized and characterized using some instrumental techniques. The TSC-NH{sub 3}-OCS adsorbent could selectively adsorb Ag(I) as AgCl{sub i}{sup 1−i} coordination anion from the Ag(I)–Cu(II)–Ni(II) simulate nickel electrolyte, especially in the case of the very high levels of Cu(II) and Ni(II), which significantly outperforms the commercial available resins. The adsorption mechanism was believed to be electrostatic interaction of the protonated bands of AgCl{sub 4}{sup 3−} with protonated thiol form of the thioamide units by FTIR and XPS analysis. The maximum adsorption capacity in the Ag(I) single and Ag(I)–Cu(II)–Ni(II) ternary system were obtained and calculated as 153.54 and 46.69 mg g{sup −1}, respectively. The reasons that the maximum adsorption capacity of AgCl{sub i}{sup 1−i} from the single and ternary system varied widely could be explained by adsorption kinetic and thermodynamic results. In addition, three successive sorption/desorption cycle runs from ternary system were performed which indicated that the TSC-NH{sub 3}-OCS adsorbent has a good performance for recovery Ag(I) from simulate nickel electrolyte.

  18. Synthesis and growth mechanism of sponge-like nickel using a hydrothermal method

    Science.gov (United States)

    Shao, Bin; Yin, Xueguo; Hua, Weidong; Ma, Yilong; Sun, Jianchun; Li, Chunhong; Chen, Dengming; Guo, Donglin; Li, Kejian

    2018-05-01

    Sponge-like nickel composed of micro-chains with a diameter of 1-5 μm was selectively synthesized by the hydrothermal method, using sodium hydroxide (NaOH) as the alkaline reagent, aqueous hydrazine as reducing agent and citric acid as a coordination agent. The time-dependent samples prepared at different NaOH concentrations were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and Fourier transform infrared spectrometer (FTIR). The results showed that the agglomerates of nickel citrate hydrazine complex nanoplates were first precipitated and then reduced to prickly nickel micro-chains at a lower NaOH concentration, which played a role in the further formation of sponge-like nickel. Also, the probable growth mechanism of the sponge-like nickel was proposed. The magnetic properties of sponge-like nickel were studied using a vibrating sample magnetometer. The sponge-like nickel exhibited a ferromagnetic behavior with a saturation magnetization value of 43.8 emu g-1 and a coercivity value of 120.7 Oe.

  19. Affinity labeling of the folate-methotrexate transporter from Leishmania donovani

    International Nuclear Information System (INIS)

    Beck, J.T.; Ullman, B.

    1989-01-01

    An affinity labeling technique has been developed to identify the folate-methotrexate transporter of Leishmania donovani promastigotes using activated derivatives of the ligands. These activated derivatives were synthesized by incubating folate and methotrexate with a 10-fold excess of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) for 10 min at ambient temperature in dimethyl sulfoxide. When intact wild-type (DI700) Leishmania donovani or preparations of their membranes were incubated with a 0.4 μM concentration of either activated [ 3 H]folate or activated [ 3 H]methotrexate, the radiolabeled ligands were covalently incorporated into a polypeptide with a molecular weight of approximately 46,000, as demonstrated by SDS-polyacrylamide gel electrophoresis. No affinity labeling of a 46,000-dalton protein was observed when equimolar concentrations of activated radiolabeled ligands were incubated with intact cells or membranes prepared from a methotrexate-resistant mutant clone of Leishmania donovani, MTXA5, that is genetically defective in folate-methotrexate transport capability. Time course studies indicated that maximal labeling of the 46,000-dalton protein occurred within 5-10 min of incubation of intact cells with activated ligand. These studies provide biochemical evidence that the folate-methotrexate transporter of Leishmania donovani can be identified in crude extracts by an affinity labeling technique and serve as a prerequisite to further analysis of the transport protein by providing a vehicle for subsequent purification of this membrane carrier. Moreover, these investigations suggest that the affinity labeling technique using EDC-activated ligands may be exploitable to analyze other cell surface binding proteins in Leishmania donovani, as well as in other organisms

  20. Oral nickel exposure may induce Type I hypersensitivity reaction in nickel-sensitized subjects.

    Science.gov (United States)

    Büyüköztürk, Suna; Gelincik, Aslı; Ünal, Derya; Demirtürk, Mustafa; Çelik, Dolay Damla; Erden, Sacide; Çolakoğlu, Bahattin; Erdem Kuruca, Serap

    2015-05-01

    Little is known about the clinical and immunological changes in the nickel allergic patients with systemic symptoms. We aimed to evaluate T helper cell responses of patients with different clinical presentations due to nickel. Patients having various allergic symptoms and positive patch test results to nickel and 20 controls underwent skin prick tests with nickel. IL-10, IL-4, IL-5 and IFN-gamma were measured in the culture supernatants of PBMC stimulated by nickel during lymphocyte proliferation test (LTT). 69 patients (56 female, mean age: 49.2 ± 13.1), 97% having nickel containing dental devices and 20 controls (8 female, mean age 34.9 ± 12.06) were evaluated. Skin prick tests with nickel were positive in 70% of the patients (pnickel. Nickel containing dental alloys and oral nickel intake seem to trigger systemic symptoms in previously nickel sensitized patients. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Prototype nickel component demonstration. Final report

    International Nuclear Information System (INIS)

    Boss, D.E.

    1994-01-01

    We have been developing a process to produce high-purity nickel structures from nickel carbonyl using chemical vapor deposition (CVD). The prototype demonstration effort had been separated into a number of independent tasks to allow Los Alamos National Laboratory (LANL) the greatest flexibility in tailoring the project to their needs. LANL selected three of the proposed tasks to be performed--Task 1- system modification and demonstration, Task 2-stainless steel mandrel trials, and Task 4-manufacturing study. Task 1 focused on converting the CVD system from a hot-wall to a cold-wall configuration and demonstrating the improved efficiency of the reactor type by depositing a 0.01-inch-thick nickel coating on a cylindrical substrate. Since stainless steel substrates were preferred because of their low α-emitter levels, Task 2 evaluated mandrel configurations which would allow removal of the nickel tube from the substrate. The manufacturing study was performed to develop strategies and system designs for manufacturing large quantities of the components needed for the Sudbury Nuetrino Observatory (SNO) program. Each of these tasks was successfully completed. During these efforts, BIRL successfully produced short lengths of 2-inch-diameter tubing and 6-inch-wide foil with levels of α-radiation emitting contaminants lower than either conventional nickel alloys or electroplated materials. We have produced both the tubing and foil using hot-substrate, cold-wall reactors and clearly demonstrated the advantages of higher precursor efficiency and deposition rate associated with this configuration. We also demonstrated a novel mandrel design which allowed easy removal of the nickel tubing and should dramatically simplify the production of 1.5-meter-long tubes in the production phase of the program

  2. Effects of Nickel, Chlorpyrifos and Their Mixture on the Dictyostelium discoideum Proteome

    Science.gov (United States)

    Boatti, Lara; Robotti, Elisa; Marengo, Emilio; Viarengo, Aldo; Marsano, Francesco

    2012-01-01

    Mixtures of chemicals can have additive, synergistic or antagonistic interactions. We investigated the effects of the exposure to nickel, the organophosphate insecticide chlorpyrifos at effect concentrations (EC) of 25% and 50% and their binary mixture (Ec25 + EC25) on Dictyostelium discoideum amoebae based on lysosomal membrane stability (LMS). We treated D. discoideum with these compounds under controlled laboratory conditions and evaluated the changes in protein levels using a two-dimensional gel electrophoresis (2DE) proteomic approach. Nickel treatment at EC25 induced changes in 14 protein spots, 12 of which were down-regulated. Treatment with nickel at EC50 resulted in changes in 15 spots, 10 of which were down-regulated. Treatment with chlorpyrifos at EC25 induced changes in six spots, all of which were down-regulated; treatment with chlorpyrifos at EC50 induced changes in 13 spots, five of which were down-regulated. The mixture corresponding to EC25 of each compound induced changes in 19 spots, 13 of which were down-regulated. The data together reveal that a different protein expression signature exists for each treatment, and that only a few proteins are modulated in multiple different treatments. For a simple binary mixture, the proteomic response does not allow for the identification of each toxicant. The protein spots that showed significant differences were identified by mass spectrometry, which revealed modulations of proteins involved in metal detoxification, stress adaptation, the oxidative stress response and other cellular processes. PMID:23443088

  3. Cloning and characterization of giant panda (Ailuropoda melanoleuca) IL-18 binding protein.

    Science.gov (United States)

    Yan, Yue; Deng, Jiabo; Niu, Lili; Wang, Qiang; Yu, Jianqiu; Shao, Huanhuan; Cao, Qinghua; Zhang, Yizheng; Tan, Xuemei

    2016-06-01

    The giant panda (Ailuropoda melanoleuca) is an endangered species. Interleukin-18 (IL-18) plays an important role in the innate and adaptive immune responses by inducing IFN-γ. IL-18 has been implicated in the pathogenesis of various diseases. IL-18 binding protein (IL-18BP) is an intrinsic inhibitor of IL-18 that possesses higher affinity to IL-18. In this study, we cloned and characterized IL-18BP in giant panda (AmIL-18BP) from the spleen. The amino acid sequence of giant panda IL-18BP ORF shared about 65% identities with other species. To evaluate the effects of AmIL-18BP on the immune responses, we expressed the recombinant AmIL-18BP in Escherichia coli BL21 (DE3).The fusing protein PET-AmIL-18BP was purified by nickel affinity column chromatography. The biological function of purified PET-AmIL-18BP was determined on mice splenocyte by qRT-PCR. The results showed that AmIL-18BP was functional and could significantly reduce IFN-γ production in murine splenocytes. These results will facilitate the study of protecting giant panda on etiology and immunology. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. A new BODIPY/nanoparticle/Ni affinity system for binding of cytochrome c

    Energy Technology Data Exchange (ETDEWEB)

    Maltas, Esra, E-mail: maltasesra@gmail.com [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Selcuk University, Faculty of Science, Department of Biochemistry, 42075 Konya (Turkey); Kursunlu, Ahmed Nuri [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Arslan, Gulsin [Selcuk University, Faculty of Science, Department of Biochemistry, 42075 Konya (Turkey); Selcuk University, Advanced Research Technology and Application Center, 42075 Konya (Turkey); Ozmen, Mustafa [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Selcuk University, Advanced Research Technology and Application Center, 42075 Konya (Turkey)

    2015-09-15

    Highlights: • BODIPY was synthesized, and then attached to magnetic nanoparticles. • Ni(II) ions were chelated on prepared material. • The binding of cytochrome c to obtained material was studied. - Abstract: In this study, 3,5-{Bis[4,4-difluoro, 8-(2,6-diethyl, 1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene)]}benzoylchloride (BODIPY) was synthesized for the improving of a new immobilized metal affinity supporting material. Firstly, the synthesized BODIPY was immobilized on iron oxide superparamagnetic nanoparticles (SPIONs) and then, Ni(II) ions were chelated with the active terminals of BODIPY on nanoparticles surfaces to prepare an immobilized metal affinity (IMA) adsorbent for protein adsorption. The amount of BODIPY coated on SPIONs was about 29.7 μM at 10 mg nanoparticles. 738 μmol of Ni(II) ions were loaded to 10 mg of the SPIONs/BODIPY. The binding amount of cytochrome c was found to be 170 μg to the SPIONs/BODIPY/Ni at pH 7.4. The binding amount of the molecules on SPIONs was analyzed by using UV–vis, fluorescence and atomic absorption spectroscopy. The characterization of the prepared surfaces was performed by FT-IR, SEM and TEM.

  5. Carbohydrate vs protein supplementation for recovery of neuromuscular function following prolonged load carriage

    Directory of Open Access Journals (Sweden)

    Fallowfield Joanne L

    2010-01-01

    Full Text Available Abstract Background This study examined the effect of carbohydrate and whey protein supplements on recovery of neuromuscular function after prolonged load carriage. Methods Ten male participants (body mass: 81.5 ± 10.5 kg, age: 28 ± 9 years, O2max: 55.0 ± 5.5 ml·kg-1·min-1 completed three treadmill walking tests (2 hr, 6.5 km·h-1, carrying a 25 kg backpack consuming 500 ml of either: (1 Placebo (flavoured water [PLA], (2 6.4% Carbohydrate Solution [CHO] or (3 7.0% Whey Protein Solution [PRO]. For three days after load carriage, participants consumed two 500 ml supplement boluses. Muscle performance was measured before and at 0, 24, 48 and 72 h after load carriage, during voluntary and electrically stimulated contractions. Results Isometric knee extension force decreased immediately after load carriage with no difference between conditions. During recovery, isometric force returned to pre-exercise values at 48 h for CHO and PRO but at 72 h for PLA. Voluntary activation decreased immediately after load carriage and returned to pre-exercise values at 24 h in all conditions (P = 0.086. During recovery, there were no differences between conditions for the change in isokinetic peak torque. Following reductions immediately after load carriage, knee extensor and flexor peak torque (60°·s-1 recovered to pre-exercise values at 72 h. Trunk extensor and flexor peak torque (15°·s-1 recovered to pre-exercise values at 24 h (P = 0.091 and 48 h (P = 0.177, respectively. Conclusion Recovery of neuromuscular function after prolonged load carriage is improved with either carbohydrate or whey protein supplementation for isometric contractions but not for isokinetic contractions.

  6. ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

    Science.gov (United States)

    Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

    2010-01-01

    Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

  7. Can we beat the biotin-avidin pair?: cucurbit[7]uril-based ultrahigh affinity host-guest complexes and their applications.

    Science.gov (United States)

    Shetty, Dinesh; Khedkar, Jayshree K; Park, Kyeng Min; Kim, Kimoon

    2015-12-07

    The design of synthetic, monovalent host-guest molecular recognition pairs is still challenging and of particular interest to inquire into the limits of the affinity that can be achieved with designed systems. In this regard, cucurbit[7]uril (CB[7]), an important member of the host family cucurbit[n]uril (CB[n], n = 5-8, 10, 14), has attracted much attention because of its ability to form ultra-stable complexes with multiple guests. The strong hydrophobic effect between the host cavity and guests, ion-dipole and dipole-dipole interactions of guests with CB portals helps in cooperative and multiple noncovalent interactions that are essential for realizing such strong complexations. These highly selective, strong yet dynamic interactions can be exploited in many applications including affinity chromatography, biomolecule immobilization, protein isolation, biological catalysis, and sensor technologies. In this review, we summarize the progress in the development of high affinity guests for CB[7], factors affecting the stability of complexes, theoretical insights, and the utility of these high affinity pairs in different challenging applications.

  8. Fed-batch production of hydrophobin RodB from Aspergillus fumigatus in host Pichia pastoris

    DEFF Research Database (Denmark)

    Pedersen, Mona Højgaard; Borodina, Irina; Frisvad, Jens Christian

    was dependent on the methanol-induced AOX1 promoter. Later production was scaled up to a 2 L fed-batch fermentor. Protein production was analyzed by SDS-PAGE, coomassie and silver-stained, as well as western blotting using an anti-his detection antibody. RodB was purified using His-select Nickel Affinity gel....... The emulsifying property of rRodB was investigated using olive oil stained with Sudan black suspended in tris-buffer. The stability of oil micelles were studied by light microscopy. Results: Protein bands of expected size were detected by SDS-PAGE and western blotting in both the fermentation broth and excess...

  9. Age-Related Changes in Selective Attention and Perceptual Load During Visual Search

    OpenAIRE

    Madden, David J.; Langley, Linda K.

    2003-01-01

    Three visual search experiments were conducted to test the hypothesis that age differences in selective attention vary as a function of perceptual load (E. A. Maylor & N. Lavie, 1998). Under resource-limited conditions (Experiments 1 and 2), the distraction from irrelevant display items generally decreased as display size (perceptual load) increased. This perceptual load effect was similar for younger and older adults, contrary to the findings of Maylor and Lavie. Distraction at low perceptua...

  10. Excessive nickel release from mobile phones--a persistent cause of nickel allergy and dermatitis

    DEFF Research Database (Denmark)

    Jensen, Peter; Johansen, Jeanne D; Zachariae, Claus

    2011-01-01

    Despite the political intention to limit nickel allergy and dermatitis in Europeans, nickel allergy remains frequent. There are several explanations for the persistence of nickel allergy and dermatitis, including the increasing use of mobile phones. Before regulation of nickel release from mobile...... phones, we showed that eight (19.5%) of 41 mobile phones marketed in Denmark between 2003 and 2007 released nickel in concentrations that may result in nickel allergy and dermatitis. In 2009, the EU Nickel Directive was revised to include nickel-releasing mobile phones....

  11. Excessive nickel release from mobile phones--a persistent cause of nickel allergy and dermatitis

    DEFF Research Database (Denmark)

    Jensen, Peter; Johansen, Jeanne D; Zachariae, Claus

    2011-01-01

    phones, we showed that eight (19.5%) of 41 mobile phones marketed in Denmark between 2003 and 2007 released nickel in concentrations that may result in nickel allergy and dermatitis. In 2009, the EU Nickel Directive was revised to include nickel-releasing mobile phones.......Despite the political intention to limit nickel allergy and dermatitis in Europeans, nickel allergy remains frequent. There are several explanations for the persistence of nickel allergy and dermatitis, including the increasing use of mobile phones. Before regulation of nickel release from mobile...

  12. Nickel exposure induces oxidative damage to mitochondrial DNA in Neuro2a cells: the neuroprotective roles of melatonin.

    Science.gov (United States)

    Xu, Shang-Cheng; He, Min-Di; Lu, Yong-Hui; Li, Li; Zhong, Min; Zhang, Yan-Wen; Wang, Yuan; Yu, Zheng-Ping; Zhou, Zhou

    2011-11-01

    Recent studies suggest that oxidative stress and mitochondrial dysfunction play important roles in the neurotoxicity of nickel. Because mitochondrial DNA (mtDNA) is highly vulnerable to oxidative stress and melatonin can efficiently protect mtDNA against oxidative damage in various pathological conditions, the aims of this study were to determine whether mtDNA oxidative damage was involved in the neurotoxicity of nickel and to assay the neuroprotective effects of melatonin in mtDNA. In this study, we exposed mouse neuroblastoma cell lines (Neuro2a) to different concentrations of nickel chloride (NiCl(2), 0.125, 0.25, and 0.5 mm) for 24 hr. We found that nickel significantly increased reactive oxygen species (ROS) production and mitochondrial superoxide levels. In addition, nickel exposure increased mitochondrial 8-hydroxyguanine (8-OHdG) content and reduced mtDNA content and mtDNA transcript levels. Consistent with this finding, nickel was found to destroy mtDNA nucleoid structure and decrease protein levels of Tfam, a key protein component for nucleoid organization. However, all the oxidative damage to mtDNA induced by nickel was efficiently attenuated by melatonin pretreatment. Our results suggest that oxidative damage to mtDNA may account for the neurotoxicity of nickel. Melatonin has great pharmacological potential in protecting mtDNA against the adverse effects of nickel in the nervous system. © 2011 John Wiley & Sons A/S.

  13. Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

    Science.gov (United States)

    Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

    2016-03-01

    Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Synthesis of Nickel and Nickel Hydroxide Nano powders by Simplified Chemical Reduction

    International Nuclear Information System (INIS)

    Tientong, J.; Garcia, S.; Thurber, C.R.; Golden, T.D.

    2014-01-01

    Nickel nano powders were synthesized by a chemical reduction of nickel ions with hydrazine hydrate at ph ∼ 12.5. Sonication of the solutions created a temperature of 54-65 °C to activate the reduction reaction of nickel nanoparticles. The solution ph affected the composition of the resulting nanoparticles. Nickel hydroxide nanoparticles were formed from an alkaline solution (ph ∼10) of nickel-hydrazine complexed by dropwise titration. X-ray diffraction of the powder and the analysis of the resulting Williamson-Hall plots revealed that the particle size of the powders ranged from 12 to 14 nm. Addition of polyvinylpyrrolidone into the synthesis decreased the nickel nanoparticle size to approximately 7 nm. Dynamic light scattering and scanning electron microscopy confirmed that the particles were in the nanometer range. The structure of the synthesized nickel and nickel hydroxide nanoparticles was identified by X-ray diffraction and Fourier transform infrared spectroscopy.

  15. Spinel formation for stabilizing simulated nickel-laden sludge with aluminum-rich ceramic precursors.

    Science.gov (United States)

    Shih, Kaimin; White, Tim; Leckie, James O

    2006-08-15

    The feasibility of stabilizing nickel-laden sludge from commonly available Al-rich ceramic precursors was investigated and accomplished with high nickel incorporation efficiency. To simulate the process, nickel oxide was mixed alternatively with gamma-alumina, corundum, kaolinite, and mullite and was sintered from 800 to 1480 degrees C. The nickel aluminate spinel (NiAl2O4) was confirmed as the stabilization phase for nickel and crystallized with efficiencies greater than 90% for all precursors above 1250 degrees C and 3-h sintering. The nickel-incorporation reaction pathways with these precursors were identified, and the microstructure and spinel yield were investigated as a function of sintering temperature with fixed sintering time. This study has demonstrated a promising process for forming nickel spinel to stabilize nickel-laden sludge from a wide range of inexpensive ceramic precursors, which may provide an avenue for economically blending waste metal sludges via the building industry processes to reduce the environmental hazards of toxic metals. The correlation of product textures and nickel incorporation efficiencies through selection of different precursors also provides the option of tailoring property-specific products.

  16. Binding affinity toward human prion protein of some anti-prion compounds - Assessment based on QSAR modeling, molecular docking and non-parametric ranking.

    Science.gov (United States)

    Kovačević, Strahinja; Karadžić, Milica; Podunavac-Kuzmanović, Sanja; Jevrić, Lidija

    2018-01-01

    The present study is based on the quantitative structure-activity relationship (QSAR) analysis of binding affinity toward human prion protein (huPrP C ) of quinacrine, pyridine dicarbonitrile, diphenylthiazole and diphenyloxazole analogs applying different linear and non-linear chemometric regression techniques, including univariate linear regression, multiple linear regression, partial least squares regression and artificial neural networks. The QSAR analysis distinguished molecular lipophilicity as an important factor that contributes to the binding affinity. Principal component analysis was used in order to reveal similarities or dissimilarities among the studied compounds. The analysis of in silico absorption, distribution, metabolism, excretion and toxicity (ADMET) parameters was conducted. The ranking of the studied analogs on the basis of their ADMET parameters was done applying the sum of ranking differences, as a relatively new chemometric method. The main aim of the study was to reveal the most important molecular features whose changes lead to the changes in the binding affinities of the studied compounds. Another point of view on the binding affinity of the most promising analogs was established by application of molecular docking analysis. The results of the molecular docking were proven to be in agreement with the experimental outcome. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Nickel concentrations in fingernails as a measure of occupational exposure to nickel

    DEFF Research Database (Denmark)

    Peters, K; Gammelgaard, Bente; Menné, T

    1991-01-01

    in nails (p less than 0.001). The difference between the 2 levels was also significant (p less than 0.001). No correlation between the nickel concentration in fingernails and the duration of exposure could be demonstrated. It was concluded that the higher the nickel level in the fingernails, the greater...... is the possibility that the person is occupationally exposed to nickel. Nail analysis is suggested as a measure of occupational exposure to nickel.......The nickel concentration in fingernails from 2 groups of people occupationally exposed to nickel was determined. In one group, comprising 83 persons moderately exposed to nickel, the mean +/- standard deviation (SD) was 29.2 micrograms/g +/- 56.7 micrograms/g and the median 13.8 micrograms/g (range...

  18. Nickel detoxification and plant growth promotion by multi metal resistant plant growth promoting Rhizobium species RL9.

    Science.gov (United States)

    Wani, Parvaze Ahmad; Khan, Mohammad Saghir

    2013-07-01

    Pollution of the biosphere by heavy metals is a global threat that has accelerated dramatically since the beginning of industrial revolution. The aim of the study is to check the resistance of RL9 towards the metals and to observe the effect of Rhizobium species on growth, pigment content, protein and nickel uptake by lentil in the presence and absence of nickel. The multi metal tolerant and plant growth promoting Rhizobium strain RL9 was isolated from the nodules of lentil. The strain not only tolerated nickel but was also tolerant o cadmium, chromium, nickel, lead, zinc and copper. The strain tolerated nickel 500 μg/mL, cadmium 300 μg/mL, chromium 400 μg/mL, lead 1,400 μg/mL, zinc 1,000 μg/mL and copper 300 μg/mL, produced good amount of indole acetic acid and was also positive for siderophore, hydrogen cyanide and ammonia. The strain RL9 was further assessed with increasing concentrations of nickel when lentil was used as a test crop. The strain RL9 significantly increased growth, nodulation, chlorophyll, leghaemoglobin, nitrogen content, seed protein and seed yield compared to plants grown in the absence of bioinoculant but amended with nickel The strain RL9 decreased uptake of nickel in lentil compared to plants grown in the absence of bio-inoculant. Due to these intrinsic abilities strain RL9 could be utilized for growth promotion as well as for the remediation of nickel in nickel contaminated soil.

  19. Improvement of the Wear Resistance of Ferrous Alloys by Electroless Plating of Nickel

    Science.gov (United States)

    Kaleicheva, J. K.; Karaguiozova, Z.

    2018-01-01

    The electroless nickel (Ni) and composite nickel - nanodiamond (Ni+DND) coatings are investigated in this study. The method EFTTOM-NICKEL for electroless nickel plating with nanosized strengthening particles (DND 4-6 nm) is applied for the coating deposition. The coatings are deposited on ferrous alloys samples. The wear resistance of the coatings is performed by friction wear tests under 50-400 MPa loading conditions - in accordance with a Polish Standard PN-83/H-04302. The microstructure observations are made by optic metallographic microscope GX41 OLIMPUS and the microhardness is determined by Vickers Method. Tests for wear resistance, thickness and microhardness measurements of the coatings without heat treatment and heat treatment are performed. The heat treatment regime is investigated with the aim to optimize the thermal process control of the coated samples without excessive tempering of the substrate material. The surface fatigue failure is determined by contact fatigue test with the purpose to establish suitable conditions for production of high performance materials.

  20. Adhesion enhancement between electroless nickel and polyester fabric by a palladium-free process

    Energy Technology Data Exchange (ETDEWEB)

    Lu Yinxiang, E-mail: yxlu@fudan.edu.cn [Department of Materials Science, Fudan University, 220 Handan Road, Shanghai 200433 (China); Xue Longlong; Li Feng [Department of Materials Science, Fudan University, 220 Handan Road, Shanghai 200433 (China)

    2011-01-15

    A new, efficient, palladium- and etchant-free process for the electroless nickel plating of poly(ethylene terephthalate) (PET) fabric has been developed. PET electroless plating can be prepared in three steps, namely: (i) the grafting of thiol group onto PET, (ii) the silver Ag{sup 0} seeding of the PET surface, and (iii) the nickel metallization using electroless plating bath. Scanning electron microscopy (SEM), energy dispersive X-ray (EDX) analysis, X-ray photoelectron spectroscopy (XPS), Raman spectrometer, X-ray diffraction (XRD), and thermogravimetric analysis (TG) were used to characterize the samples in the process, and the nickel loading was quantified by weighing. This process successfully compares with the traditional one based on KMnO{sub 4}/H{sub 2}SO{sub 4} etching and palladium-based seed layer. The nickel coating obtained in