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Sample records for copper protein probed

  1. Utilizing NMR and EPR spectroscopy to probe the role of copper in prion diseases

    KAUST Repository

    Emwas, Abdul-Hamid M.

    2013-02-24

    Copper is an essential nutrient for the normal development of the brain and nervous system, although the hallmark of several neurological diseases is a change in copper concentrations in the brain and central nervous system. Prion protein (PrP) is a copper-binding, cell-surface glycoprotein that exists in two alternatively folded conformations: a normal isoform (PrPC) and a disease-associated isoform (PrPSc). Prion diseases are a group of lethal neurodegenerative disorders that develop as a result of conformational conversion of PrPC into PrPSc. The pathogenic mechanism that triggers this conformational transformation with the subsequent development of prion diseases remains unclear. It has, however, been shown repeatedly that copper plays a significant functional role in the conformational conversion of prion proteins. In this review, we focus on current research that seeks to clarify the conformational changes associated with prion diseases and the role of copper in this mechanism, with emphasis on the latest applications of NMR and EPR spectroscopy to probe the interactions of copper with prion proteins. Copyright © 2013 John Wiley & Sons, Ltd.

  2. A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

    Directory of Open Access Journals (Sweden)

    Choveaux David L

    2012-11-01

    Full Text Available Abstract Background Copper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated. Methods PlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy. Results Six P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369, containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein. Conclusion Plasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds.

  3. Copper delivery to chloroplast proteins and its regulation

    Directory of Open Access Journals (Sweden)

    Guadalupe eAguirre

    2016-01-01

    Full Text Available Copper is required for photosynthesis in chloroplasts of plants because it is a cofactor of plastocyanin, an essential electron carrier in the thylakoid lumen. Other chloroplast copper proteins are copper/zinc superoxide dismutase and polyphenol oxidase,¬ but these proteins seem to be dispensable under conditions of low copper supply when transcripts for these proteins undergo microRNA-mediated down regulation. Two ATP-driven copper transporters function in tandem to deliver copper to chloroplast compartments. This review seeks to summarize the mechanisms of copper delivery to chloroplast proteins and its regulation. We also delineate some of the unanswered questions that still remain in this field.

  4. Complexation between the fungicide tebuconazole and copper(II) probed by electrospray ionization mass spectrometry.

    Science.gov (United States)

    Dytrtová, Jana Jaklová; Jakl, Michal; Schröder, Detlef; Čadková, Eva; Komárek, Michael

    2011-04-30

    Electrospray ionization mass spectrometry (ESI-MS) is used to probe the complex formation between tebuconazole (1) and copper(II) salts, which both are commonly used fungicides in agriculture. Experiments with model solutions containing 1 and CuCl(2) reveal the initial formation of the copper(II) species [(1)CuCl](+) and [(1)(2)CuCl](+) which undergo reduction to the corresponding copper(I) ions [(1)Cu](+) and [(1)(2)Cu](+) under more drastic ionization conditions in the ESI source. In additional experiments, copper/tebuconazole complexes were also detected in samples made from soil solutions of various origin and different amount of mineralization. The direct sampling of such solutions via ESI-MS is thus potentially useful for understanding of the interactions between copper(II) salts and tebuconazole in environmental samples. Copyright © 2011 John Wiley & Sons, Ltd.

  5. Site-Specific Infrared Probes of Proteins

    Science.gov (United States)

    Ma, Jianqiang; Pazos, Ileana M.; Zhang, Wenkai; Culik, Robert M.; Gai, Feng

    2015-04-01

    Infrared spectroscopy has played an instrumental role in the study of a wide variety of biological questions. However, in many cases, it is impossible or difficult to rely on the intrinsic vibrational modes of biological molecules of interest, such as proteins, to reveal structural and environmental information in a site-specific manner. To overcome this limitation, investigators have dedicated many recent efforts to the development and application of various extrinsic vibrational probes that can be incorporated into biological molecules and used to site-specifically interrogate their structural or environmental properties. In this review, we highlight recent advancements in this rapidly growing research area.

  6. Site-Specific Infrared Probes of Proteins

    Science.gov (United States)

    Ma, Jianqiang; Pazos, Ileana M.; Zhang, Wenkai; Culik, Robert M.; Gai, Feng

    2015-01-01

    Infrared spectroscopy has played an instrumental role in studying a wide variety of biological questions. However, in many cases it is impossible or difficult to rely on the intrinsic vibrational modes of biological molecules of interest, such as proteins, to reveal structural and/or environmental information in a site-specific manner. To overcome this limitation, many recent efforts have been dedicated to the development and application of various extrinsic vibrational probes that can be incorporated into biological molecules and used to site-specifically interrogate their structural and/or environmental properties. In this Review, we highlight some recent advancements of this rapidly growing research area. PMID:25580624

  7. Functionally specified protein signatures distinctive for each of the different blue copper proteins

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    Anishetty Sharmila

    2004-09-01

    Full Text Available Abstract Background Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands. Results Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership. The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes. ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides. The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids

  8. Simultaneous Platinum and Copper Ion Attachment to a Human Copper Chaperone Protein

    Science.gov (United States)

    Hodak, Miroslav; Cvitkovic, John; Yu, Corey; Dmitriev, Oleg; Kaminski, George; Bernholc, Jerry

    2015-03-01

    Cisplatin is a potent anti-cancer drug based on a platinum ion. However, its effectiveness is decreased by cellular resistance, which involves cisplatin attaching to copper transport proteins. One of such proteins is Atox1, where cisplatin attaches to the copper binding site. Surprisingly, it was shown that both cisplatin and copper can attach to Atox1 at the same time. To study this double metal ion attachment, we use the KS/FD DFT method, which combines Kohn-Sham DFT with frozen-density DFT to achieve efficient quantum-mechanical description of explicit solvent. Calculations have so far investigated copper ion attachment to CXXC motifs present in Atox1. The addition of the platinum ion and the competition between the two metals is currently being studied. These calculations start from a molecular mechanics (MM) structural model, in which glutathione groups provide additional ligands to the Pt ion. Our goals are to identify possible Cu-Pt structures and to determine whether copper/platinum attachment is competitive, independent, or cooperative. Results will be compared to the 1H, N1 5 -HSQC NMR experiments, in which binding of copper and cisplatin to Atox1 produces distinct secondary chemical shift signatures, allowing for kinetic studies of simultaneous metal binding.

  9. Placenta Copper Transport Proteins in Preeclampsia

    Science.gov (United States)

    Placental insufficiency underlying preeclampsia (PE) is associated with impaired placental angiogenesis. As copper (Cu) is essential to angiogenesis, we investigated differences in the expression of placental Cu transporters Menkes (ATP7A), Wilsons (ATP7B) and the Cu chaperone (CCS) for superoxide d...

  10. Atom probe tomography characterization of thin copper layers on aluminum deposited by galvanic displacement.

    Science.gov (United States)

    Zhang, Yi; Ai, Jiahe; Hillier, Andrew C; Hebert, Kurt R

    2012-01-24

    ″Ultrathin″ metallization layers on the order of nanometers in thickness are increasingly used in semiconductor interconnects and other nanostructures. Aqueous deposition methods are attractive methods to produce such layers due to their low cost, but formation of ultrathin layers has proven challenging, particularly on oxide-coated substrates. This work focused on the formation of thin copper layers on aluminum, by galvanic displacement from alkaline aqueous solutions. Analysis by atom probe tomography (APT) showed that continuous copper films of approximately 1 nm thickness were formed, apparently the first demonstration of deposition of ultrathin metal layers on oxidized substrates from aqueous solutions. The APT reconstructions indicate that deposited copper replaced a portion of the surface oxide film on aluminum. The results are consistent with mechanisms in which surface hydride species on aluminum mediate deposition, either by directly reducing cupric ions or by inducing electronic conduction in the oxide, thus enabling cupric ion reduction by Al metal.

  11. Microstructural evolution in tungsten and copper probes under hydrogen irradiation at ISTTOK

    International Nuclear Information System (INIS)

    Nunes, D.; Mateus, R.; Nogueira, I.D.; Carvalho, P.A.; Correia, J.B.; Shohoji, N.; Gomes, R.B.; Fernandes, H.; Silva, C.; Franco, N.; Alves, E.

    2009-01-01

    Commercially pure tungsten and copper wires acting as Langmuir probes to estimate edge parameters of ISTTOK plasma have been investigated for long term hydrogen migration. The microstructure of both materials revealed recrystallization and strong grain growth at the most severely exposed regions. A low number of large bubbles was observed at the most severely exposed regions, whereas a high density of small intergranular bubbles was found at more moderately exposed regions. Bubble distribution, lattice parameter, grain size, Young's modulus and microhardness were assessed across longitudinal sections of the probes. The results indicate that bubble formation in tungsten and copper first wall components can be expected to occur and strategies for minimization of this retention phenomenon need to be implemented.

  12. Protein recognition by a pattern-generating fluorescent molecular probe

    Science.gov (United States)

    Pode, Zohar; Peri-Naor, Ronny; Georgeson, Joseph M.; Ilani, Tal; Kiss, Vladimir; Unger, Tamar; Markus, Barak; Barr, Haim M.; Motiei, Leila; Margulies, David

    2017-12-01

    Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.

  13. Electrically continuous graphene from single crystal copper verified by terahertz conductance spectroscopy and micro four-point probe

    DEFF Research Database (Denmark)

    Buron, Jonas Christian Due; Pizzocchero, Filippo; Jessen, Bjarke Sørensen

    2014-01-01

    . Micro four-point probe resistance values measured on graphene grown on single crystalline copper in two different voltage-current configurations show close agreement with the expected distributions for a continuous 2D conductor, in contrast with previous observations on graphene grown on commercial...... noninvasive conductance characterization methods: ultrabroadband terahertz time-domain spectroscopy and micro four-point probe, which probe the electrical properties of the graphene film on different length scales, 100 nm and 10 μm, respectively. Ultrabroadband terahertz time-domain spectroscopy allows...... copper foil. The terahertz and micro four-point probe conductance values of the graphene grown on single crystalline copper shows a close to unity correlation, in contrast with those of the graphene grown on commercial copper foil, which we explain by the absence of extended defects on the microscale...

  14. Copper and the Prion Protein: Methods, Structures, Function, and Disease

    Science.gov (United States)

    Millhauser, Glenn L.

    2007-05-01

    The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrPC to PrPSc. Examples of the TSEs include mad cow disease, chronic wasting disease in deer and elk, scrapie in goats and sheep, and kuru and Creutzfeldt-Jakob disease in humans. Although the precise function of PrPC in healthy tissues is not known, recent research demonstrates that it binds Cu(II) in an unusual and highly conserved region of the protein termed the octarepeat domain. This review describes recent connections between copper and PrPC, with an emphasis on the electron paramagnetic resonance elucidation of the specific copper-binding sites, insights into PrPC function, and emerging connections between copper and prion disease.

  15. Cloning and expression analysis of a blue copper- binding protein ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... Full Length Research Paper. Cloning and expression analysis of a blue copper- binding protein gene from Dasypyrum Villosum. Huagang He1*, Shanying Zhu1, Wenbing Wang1, Tongde Bie2 and Peidu Chen3. 1Jiangsu University. Zhenjiang 212013, P. R. China. 2Yangzhou Academy of Agricultural ...

  16. Synthesis and application of a highly selective copper ions fluorescent probe based on the coumarin group

    Science.gov (United States)

    He, Guangjie; Liu, Xiangli; Xu, Jinhe; Ji, Liguo; Yang, Linlin; Fan, Aiying; Wang, Songjun; Wang, Qingzhi

    2018-02-01

    A highly selective copper ions fluorescent probe based on the coumarin-type Schiff base derivative 1 (probe) was produced by condensation reaction between coumarin carbohydrazide and 1H-indazole-3-carbaldehyde. The UV-vis spectroscopy showed that the maximum absorption peak of compound 1 appeared at 439 nm. In the presence of Cu2 + ions, the maximum peak decreased remarkably compared with other physiological important metal ions and a new absorption peak at 500 nm appeared. The job's plot experiments showed that complexes of 1:2 binding mode were formed in CH3CN:HEPES (3:2, v/v) solution. Compound 1 exhibited a strong blue fluorescence. Upon addition of copper ions, the fluorescence gradually decreased and reached a plateau with the fluorescence quenching rate up to 98.73%. The detection limit for Cu2 + ions was estimated to 0.384 ppm. Fluorescent microscopy experiments demonstrated that probe 1 had potential to be used to investigate biological processes involving Cu2 + ions within living cells.

  17. Atom probe tomography characterizations of high nickel, low copper surveillance RPV welds irradiated to high fluences

    Science.gov (United States)

    Miller, M. K.; Powers, K. A.; Nanstad, R. K.; Efsing, P.

    2013-06-01

    The Ringhals Units 3 and 4 reactors in Sweden are pressurized water reactors (PWRs) designed and supplied by Westinghouse Electric Company, with commercial operation in 1981 and 1983, respectively. The reactor pressure vessels (RPVs) for both reactors were fabricated with ring forgings of SA 508 class 2 steel. Surveillance blocks for both units were fabricated using the same weld wire heat, welding procedures, and base metals used for the RPVs. The primary interest in these weld metals is because they have very high nickel contents, with 1.58 and 1.66 wt.% for Unit 3 and Unit 4, respectively. The nickel content in Unit 4 is the highest reported nickel content for any Westinghouse PWR. Although both welds contain less than 0.10 wt.% copper, the weld metals have exhibited high irradiation-induced Charpy 41-J transition temperature shifts in surveillance testing. The Charpy impact 41-J shifts and corresponding fluences are 192 °C at 5.0 × 1023 n/m2 (>1 MeV) for Unit 3 and 162 °C at 6.0 × 1023 n/m2 (>1 MeV) for Unit 4. These relatively low-copper, high-nickel, radiation-sensitive welds relate to the issue of so-called late-blooming nickel-manganese-silicon phases. Atom probe tomography measurements have revealed ˜2 nm-diameter irradiation-induced precipitates containing manganese, nickel, and silicon, with phosphorus evident in some of the precipitates. However, only a relatively few number of copper atoms are contained within the precipitates. The larger increase in the transition temperature shift in the higher copper weld metal from the Ringhals R3 Unit is associated with copper-enriched regions within the manganese-nickel-silicon-enriched precipitates rather than changes in their size or number density.

  18. COPPER FRACTIONATION IN PROTEINS FROM PLASMA, MUSCLE AND LIVER OF NILE TILAPIA

    OpenAIRE

    Neves, RCF; Lima, PM; Baldassini, WA; Santos, FA; Moraes, PM; Castro, GR; Padilha, PM

    2012-01-01

    COPPER FRACTIONATION IN PROTEINS FROM PLASMA, MUSCLE AND LIVER OF NILE TILAPIA. Copper fractionation in plasma, muscle and liver of Nile tilapia was performed after protein separation by 2D-PAGE. SR XRF analysis indicated the presence of copper in three protein spots of plasma, and in two protein spots of muscle and liver, respectively. Copper ions were found to be distributed mostly in proteins that had a molar mass of less than 54 kDa and greater than 13 kat and a pI in the 5.3-9.3 range. T...

  19. Serum Copper and Plasma Protein Status in Normal Pregnancy

    Directory of Open Access Journals (Sweden)

    Nushrat Noor, Nasim Jahan, Nayma Sultana

    2012-12-01

    Full Text Available AbstractBackground: Gradual alteration of serum copper and some plasma protein levels may occur with advancement of pregnancy, which is associated with increased maternal and infant morbidity and mortality.Objective: To observe serum copper and plasma protein levels in normal pregnant women of different trimesters in order to find out their nutritional status.Methods: This cross sectional study was carried out in the Department of Physiology, Sir Salimullah Medical College (SSMC, Dhaka, between 1st January 2010 and December 2010. Ninety normal pregnant women of different trimesters with age 20-30 years were included in the study group. They were selected from Out Patient Department of Obstetrics and Gynaecology, SSMC. Age matched 30 non-pregnant women were taken as control. Serum copper level was measured by Spectrophotometric method, serum total protein and albumin levels were estimated by standard method. Statistical analysis was done by one way ANOVA, Bonferroni and Pearson’s correlation coefficient test as applicable.Results: Serum Cu levels were significantly higher in all trimesters of pregnant women compared to control. Again, this value was significantly higher in 3rd trimester than that of in 1st and 2nd trimester and also in 2nd trimester than that of in 1st trimester. In addition, mean serum total protein level was significantly lower in 3rd trimester than control but no statistically significant difference was observed among different trimesters. Again, mean serum albumin level was significantly lower in 2nd and 3rd trimester than 1st trimester and control. In addition, serum Cu concentration showed significant positive correlation with different trimesters of gestation.Conclusion: This study reveals that hypercupremia along with hypoproteinemia occur in pregnant women from 1st to 3rd trimester of gestation. This gradual alteration of micro and macronutrients become more profound with advancement of pregnancy.

  20. Probing nanoparticle effect in protein-surfactant complexes

    Science.gov (United States)

    Mehan, Sumit; Aswal, V. K.; Kohlbrecher, J.

    2015-06-01

    SANS experiments have been carried to probe the role of anionic silica nanoparticles in the anionic BSA protein-cationic DTAB surfactant complexes. In protein-surfactant complex, surfactant molecules aggregate to form micelle-like clusters along the unfolded polypeptide chains of the protein. The nanoparticle aggregation mediated by oppositely charged protein-surfactant complex coexists with the free protein-surfactant complexes in the nanoparticle-protein-surfactant system. There is rearrangement of micelles in adsorbed protein-surfactant complex on nanoparticles in leading to their (nanoparticle) aggregation. On the other hand, the unfolding of protein in free protein-surfactant complex is found to be significantly enhanced in presence of nanoparticles.

  1. Determination of the Electron Self-Exchange Rates of Blue Copper Proteins by Super-WEFT NMR Spectroscopy

    DEFF Research Database (Denmark)

    Ma, Lixin; Philipp, Else Astrid; Led, Jens J.

    2001-01-01

    Anabaena variabilis plastocyanin, blue copper proteins, electron self-exchange rates, electron transfer, super-WEFT NMR......Anabaena variabilis plastocyanin, blue copper proteins, electron self-exchange rates, electron transfer, super-WEFT NMR...

  2. Direct electrochemistry of blue copper proteins at boron-doped diamond electrodes

    Energy Technology Data Exchange (ETDEWEB)

    McEvoy, James P. [Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA (United Kingdom); Foord, John S. [Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA (United Kingdom)]. E-mail: john.foord@chem.ox.ac.uk

    2005-05-05

    Boron-doped diamond (BDD) is a promising electrode material for use in the spectro-electrochemical study of redox proteins and, in this investigation, cyclic voltammetry was used to obtain quasi-reversible electrochemical responses from two blue copper proteins, parsley plastocyanin and azurin from Pseudomonas aeruginosa. No voltammetry was observed at the virgin electrodes, but signals were observed if the electrodes were anodised, or abraded with alumina, prior to use. Plastocyanin, which has a considerable overall negative charge and a surface acidic patch which is important in forming a productive electron transfer complex with its redox partners, gave a faradaic signal at pre-treated BDD only in the presence of neomycin, a positively charged polyamine. The voltammetry of azurin, which has a small overall charge and no surface acidic patch, was obtained identically in the presence and absence of neomycin. Investigations were also carried out into the voltammetry of two site-directed mutants of azurin, M64E azurin and M44K azurin, each of which introduce a charge into the protein's surface hydrophobic patch. The oxidizing and cleaning effects of the BDD electrode pre-treatments were studied electrochemically using two inorganic probe ions, Fe(China){sub 6} {sup 3-} and Ru(NH{sub 3}){sub 6} {sup 3+}, and by X-ray photoelectron spectroscopy (XPS). All of the electrochemical results are discussed in relation to the electrostatic and hydrophobic contributions to the protein/diamond electrochemical interaction.

  3. Phthalic Acid Chemical Probes Synthesized for Protein-Protein Interaction Analysis

    Directory of Open Access Journals (Sweden)

    Chin-Jen Wu

    2013-06-01

    Full Text Available Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP. According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES was deposited on silicon dioxides (SiO2 particles and phthalate chemical probes were manufactured from phthalic acid and APTES–SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA software showed that these chemical probes were a practical technique for protein-protein interaction analysis.

  4. Preferred sites and pathways for electron transfer in blue copper proteins

    DEFF Research Database (Denmark)

    Farver, O; Pecht, I

    1988-01-01

    of where and how electrons are transferred to and from the copper-ion have been investigated. One experimental approach developed in order to pursue these problems is that of reductively labeling several representative, yet structurally distinct blue single copper proteins; azurin, plastocyanin......, and stellacyanin with chromium ions. In all three cases, a substitution inert Cr(III)-adduct is formed when the oxidized protein is reduced by Cr(II)ag ions. In azurin, Cr(III) binds to the Glu-91 carboxylate approximately 10 A from the copper center. In both plastocyanin and stellacyanin the Cr(III) label is most...... probably also coordinated to carboxylate groups, present in plastocyanin, and in stellacyanin 12 A and 6 A, respectively, from the copper center. The salient feature emerging from examination of the three copper proteins is that a pi-facilitated electron transfer (E.T.) pathway may be operative; in azurin...

  5. Computational probing protein-protein interactions targeting small molecules.

    Science.gov (United States)

    Wang, Yong-Cui; Chen, Shi-Long; Deng, Nai-Yang; Wang, Yong

    2016-01-15

    With the booming of interactome studies, a lot of interactions can be measured in a high throughput way and large scale datasets are available. It is becoming apparent that many different types of interactions can be potential drug targets. Compared with inhibition of a single protein, inhibition of protein-protein interaction (PPI) is promising to improve the specificity with fewer adverse side-effects. Also it greatly broadens the drug target search space, which makes the drug target discovery difficult. Computational methods are highly desired to efficiently provide candidates for further experiments and hold the promise to greatly accelerate the discovery of novel drug targets. Here, we propose a machine learning method to predict PPI targets in a genomic-wide scale. Specifically, we develop a computational method, named as PrePPItar, to Predict PPIs as drug targets by uncovering the potential associations between drugs and PPIs. First, we survey the databases and manually construct a gold-standard positive dataset for drug and PPI interactions. This effort leads to a dataset with 227 associations among 63 PPIs and 113 FDA-approved drugs and allows us to build models to learn the association rules from the data. Second, we characterize drugs by profiling in chemical structure, drug ATC-code annotation, and side-effect space and represent PPI similarity by a symmetrical S-kernel based on protein amino acid sequence. Then the drugs and PPIs are correlated by Kronecker product kernel. Finally, a support vector machine (SVM), is trained to predict novel associations between drugs and PPIs. We validate our PrePPItar method on the well-established gold-standard dataset by cross-validation. We find that all chemical structure, drug ATC-code, and side-effect information are predictive for PPI target. Moreover, we can increase the PPI target prediction coverage by integrating multiple data sources. Follow-up database search and pathway analysis indicate that our new

  6. A Molecular Mechanism for Copper Transportation to Tyrosinase That Is Assisted by a Metallochaperone, Caddie Protein*

    Science.gov (United States)

    Matoba, Yasuyuki; Bando, Naohiko; Oda, Kosuke; Noda, Masafumi; Higashikawa, Fumiko; Kumagai, Takanori; Sugiyama, Masanori

    2011-01-01

    The Cu(II)-soaked crystal structure of tyrosinase that is present in a complex with a protein, designated “caddie,” which we previously determined, possesses two copper ions at its catalytic center. We had identified two copper-binding sites in the caddie protein and speculated that copper bound to caddie may be transported to the tyrosinase catalytic center. In our present study, at a 1.16–1.58 Å resolution, we determined the crystal structures of tyrosinase complexed with caddie prepared by altering the soaking time of the copper ion and the structures of tyrosinase complexed with different caddie mutants that display little or no capacity to activate tyrosinase. Based on these structures, we propose a molecular mechanism by which two copper ions are transported to the tyrosinase catalytic center with the assistance of caddie acting as a metallochaperone. PMID:21730070

  7. The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells

    Energy Technology Data Exchange (ETDEWEB)

    Gough, Mallory, E-mail: m.gough1@lancaster.ac.uk; Blanthorn-Hazell, Sophee, E-mail: s.blanthorn-hazell@lancaster.ac.uk; Delury, Craig, E-mail: c.delury@lancaster.ac.uk; Parkin, Edward, E-mail: e.parkin@lancaster.ac.uk

    2014-10-31

    Highlights: • Copper levels are elevated in the tumour microenvironment. • APP mitigates copper-induced growth inhibition of DU145 prostate cancer (PCa) cells. • The APP intracellular domain is a prerequisite; soluble forms have no effect. • The E1 CuBD of APP is also a prerequisite. • APP copper binding potentially mitigates copper-induced PCa cell growth inhibition. - Abstract: Copper plays an important role in the aetiology and growth of tumours and levels of the metal are increased in the serum and tumour tissue of patients affected by a range of cancers including prostate cancer (PCa). The molecular mechanisms that enable cancer cells to proliferate in the presence of elevated copper levels are, therefore, of key importance in our understanding of tumour growth progression. In the current study, we have examined the role played by the amyloid precursor protein (APP) in mitigating copper-induced growth inhibition of the PCa cell line, DU145. A range of APP molecular constructs were stably over-expressed in DU145 cells and their effects on cell proliferation in the presence of copper were monitored. Our results show that endogenous APP expression was induced by sub-toxic copper concentrations in DU145 cells and over-expression of the wild-type protein was able to mitigate copper-induced growth inhibition via a mechanism involving the cytosolic and E1 copper binding domains of the full-length protein. APP likely represents one of a range of copper binding proteins that PCa cells employ in order to ensure efficient proliferation despite elevated concentrations of the metal within the tumour microenvironment. Targeting the expression of such proteins may contribute to therapeutic strategies for the treatment of cancers.

  8. Combined gel filtration, biuret/copper method compared with an immunochemical method for urinary protein measurement.

    Science.gov (United States)

    Peele, J D; Gadsden, R H; Loadholt, C B

    1977-01-01

    We compared an immunochemical method specific for plasma proteins with a chemical method, in which interfering substances are separated by gel filtration and "total" urinary protein is determined by the biuret reaction followed by reaction of protein-bound copper with diethyldithiocarbamate after a second gel filtration to remove nonprotein-bound copper. More than 250 24-h urine samples were analyzed by each method. There was linear agreement and a correlation of 0.96 between the two methods, but urinary protein values determined by the immunochemical method, especially for patients with multiple myeloma, were lower than by the chemical method.

  9. Probing hydrogen bonding interactions and proton transfer in proteins

    Science.gov (United States)

    Nie, Beining

    Scope and method of study. Hydrogen bonding is a fundamental element in protein structure and function. Breaking a single hydrogen bond may impair the stability of a protein. It is therefore important to probe dynamic changes in hydrogen bonding interactions during protein folding and function. Time-resolved Fourier transform infrared spectroscopy is highly sensitive to hydrogen bonding interactions. However, it lacks quantitative correlation between the vibrational frequencies and the number, type, and strength of hydrogen bonding interactions of ionizable and polar residues. We employ quantum physics theory based ab initio calculations to study the effects of hydrogen bonding interactions on vibrational frequencies of Asp, Glu, and Tyr residues and to develop vibrational spectral markers for probing hydrogen bonding interactions using infrared spectroscopy. In addition, proton transfer process plays a crucial role in a wide range of energy transduction, signal transduction, and enzymatic reactions. We study the structural basis for proton transfer using photoactive yellow protein as an excellent model system. Molecular dynamics simulation is employed to investigate the structures of early intermediate states. Quantum theory based ab initio calculations are used to study the impact of hydrogen bond interactions on proton affinity and proton transfer. Findings and conclusions. Our extensive density function theory based calculations provide rich structural, spectral, and energetic information on hydrogen bonding properties of protonated side chain groups of Asp/Glu and Tyr. We developed vibrational spectral markers and 2D FTIR spectroscopy for structural characterization on the number and the type of hydrogen bonding interactions of the COOH group of Asp/Glu and neutral phenolic group of Tyr. These developments greatly enhance the power of time-resolved FTIR spectroscopy as a major experimental tool for structural characterization of functionally important

  10. The Synthesis of a Coumarin Carbohydrazide Dinuclear Copper Complex Based Fluorescence Probe and Its Detection of Thiols.

    Directory of Open Access Journals (Sweden)

    Guangjie He

    Full Text Available Small-molecule thiols, such as cysteine (CYS and glutathione (GSH, are essential for maintaining the cellular redox environment and play important roles in regulating various cellular physiological functions. A fluorescence probe (compound 1-Cu2+ for thiols based on coumarin carbohydrazide dinuclear copper complex was developed. Compound 1 was synthesized from the reaction of 7-(diethylamino-2-oxo-2H-chromene-3-carbohydrazide with 4-tert-butyl-2,6- diformylphenol. Accordingly, the copper complex (compound 1-Cu2+ was prepared by mixing compound 1 with 2 equivalents copper ions. Compound 1 had strong fluorescence while compound 1-Cu2+ hardly possessed fluorescence owing to the quenching nature of paramagnetism Cu2+ to the fluorescence molecule excited state. However, the fluorescence intensity of compound 1-Cu2+ was increased dramatically after the addition of thiol-containing amino acids, but not the other non-sulfhydryl amino acids. UV-vis absorption and fluorescence spectra indicated that compound 1-Cu2+ had good selectivity and sensitivity for thiols such as glutathione in CH3CN:H2O (3:2, v/v PBS solution. The fluorescence imaging experiments implied that compound 1-Cu2+ has potential application in thiol-containing amino acids detection in living cells.

  11. Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein

    DEFF Research Database (Denmark)

    Jensen, P Y; Bonander, N; Møller, L B

    1999-01-01

    spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two...... and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains...... is cooperative, with Hill coefficients of 1.5 and 4, respectively. The apparent affinities are described by K(0.5), determined to be 65 microM and 19 microM for constructs containing two and six domains, respectively. Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration...

  12. Functional understanding of the versatile protein copper metabolism MURR1 domain 1 (COMMD1) in copper homeostasis

    NARCIS (Netherlands)

    Fedoseienko, Alina; Bartuzi, Paulina; van de Sluis, Bart

    2014-01-01

    Copper is an important cofactor in numerous biological processes in all living organisms. However, excessive copper can be extremely toxic, so it is vital that the copper level within a cell is tightly regulated. The damaging effect of copper is seen in several hereditary forms of copper toxicity in

  13. Probing the Intramolecular Metal-Selenoether Interaction in a Bis(iminosemiquinone) copper(II) Compound

    Czech Academy of Sciences Publication Activity Database

    Bubrin, M.; Paretzki, A.; Hübner, R.; Beyer, K.; Schwederski, B.; Neugebauer, P.; Záliš, Stanislav; Kaim, W.

    2017-01-01

    Roč. 643, č. 21 (2017), s. 1621-1627 ISSN 0044-2313 R&D Projects: GA MŠk(CZ) LTC17052 Institutional support: RVO:61388955 Keywords : EPR spectroscopy * Molecular structure * Selenium ligand * Spectroelectrochemistry * Copper Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 1.144, year: 2016

  14. Complexation between the fungicide tebuconazole and copper(II) probed by electrospray ionization mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Jaklová Dytrtová, Jana; Jakl, M.; Schröder, Detlef; Čadková, E.; Komárek, M.

    2011-01-01

    Roč. 25, č. 8 (2011), s. 1037-1042 ISSN 0951-4198 Institutional research plan: CEZ:AV0Z40550506 Keywords : copper * electrospray ionization * mass spectrometry * tebuconazole * soil solutions Subject RIV: CC - Organic Chemistry Impact factor: 2.790, year: 2011

  15. Ergothioneine prevents copper-induced oxidative damage to DNA and protein by forming a redox-inactive ergothioneine-copper complex.

    Science.gov (United States)

    Zhu, Ben-Zhan; Mao, Li; Fan, Rui-Mei; Zhu, Jun-Ge; Zhang, Ying-Nan; Wang, Jing; Kalyanaraman, Balaraman; Frei, Balz

    2011-01-14

    Ergothioneine (2-mercaptohistidine trimethylbetaine) is a naturally occurring amino acid analogue found in up to millimolar concentrations in several tissues and biological fluids. However, the biological functions of ergothioneine remain incompletely understood. In this study, we investigated the role of ergothioneine in copper-induced oxidative damage to DNA and protein, using two copper-containing systems: Cu(II) with ascorbate and Cu(II) with H(2)O(2) [0.1 mM Cu(II), 1 mM ascorbate, and 1 mM H(2)O(2)]. Oxidative damage to DNA and bovine serum albumin was measured as strand breakage and protein carbonyl formation, respectively. Ergothioneine (0.1-1.0 mM) provided strong, dose-dependent protection against oxidation of DNA and protein in both copper-containing systems. In contrast, only limited protection was observed with the purported hydroxyl radical scavengers, dimethyl sulfoxide and mannitol, even at concentrations as high as 100 mM. Ergothioneine also significantly inhibited copper-catalyzed oxidation of ascorbate and competed effectively with histidine and 1,10-phenanthroline for binding of cuprous copper, but not cupric copper, as demonstrated by UV-visible and low-temperature electron spin resonance techniques. We conclude that ergothioneine is a potent, natural sulfur-containing antioxidant that prevents copper-dependent oxidative damage to biological macromolecules by forming a redox-inactive ergothioneine-copper complex.

  16. A large volume flat coil probe for oriented membrane proteins

    Science.gov (United States)

    Gor'kov, Peter L.; Chekmenev, Eduard Y.; Fu, Riqiang; Hu, Jun; Cross, Timothy A.; Cotten, Myriam; Brey, William W.

    2006-07-01

    15N detection of mechanically aligned membrane proteins benefits from large sample volumes that compensate for the low sensitivity of the observe nuclei, dilute sample preparation, and for the poor filling factor arising from the presence of alignment plates. Use of larger multi-tuned solenoids, however, is limited by wavelength effects that lead to inhomogeneous RF fields across the sample, complicating cross-polarization experiments. We describe a 600 MHz 15N-1H solid-state NMR probe with large (580 mm3) RF solenoid for high-power, multi-pulse sequence experiments, such as polarization inversion spin exchange at the magic angle (PISEMA). In order to provide efficient detection for 15N, a 4-turn solenoidal sample coil is used that exceeds 0.27λ at the 600 MHz 1H resonance. A balanced tuning-matching circuit is employed to preserve RF homogeneity across the sample for adequate magnetization transfer from 1H to 15N. We describe a procedure for optimization of the shorted 1/4λ coaxial trap that allows for the sufficiently strong RF fields in both 1H and 15N channels to be achieved within the power limits of 300 W 1H and 1 kW 15N amplifiers. The 8 × 6 × 12 mm solenoid sustains simultaneous B1 irradiation of 100 kHz at 1H frequency and 51 kHz at 15N frequency for at least 5 ms with 265 and 700 W of input power in the respective channels. The probe functionality is demonstrated by 2D 15N-1H PISEMA spectroscopy for two applications at 600 MHz.

  17. Isotopic probing of molecular oxygen activation at copper(I) sites.

    Science.gov (United States)

    Lanci, Michael P; Smirnov, Valeriy V; Cramer, Christopher J; Gauchenova, Ekaterina V; Sundermeyer, Jörg; Roth, Justine P

    2007-11-28

    Copper-dioxygen (CuO2) adducts are frequently proposed as intermediates in enzymes, yet their electronic and vibrational structures have not always been understood. [Cu(eta1-O2)TMG3tren]+ (TMG3tren = 1,1,1-tris{2-[N2-(1,1,3,3-tetramethylguanidino)]ethyl}amine) features end-on (eta1) O2 coordination in the solid state. Described here is an investigation of the compound's solution properties by nuclear magnetic resonance spectroscopy, density functional calculations, and oxygen isotope effects. The study yields two major findings. First, [Cu(eta1-O2)TMG3tren]+ is paramagnetic due to a triplet electronic structure; this is in contrast to other copper compounds where O2 is bound in a side-on manner. Second, the oxygen equilibrium isotope effect upon O2 binding to copper(I) (18O EIE [triple bond] K(16O16O)/K(16O18O) = 1.0148 +/- 0.0012) is significantly larger than those determined for iron and cobalt eta1-O2 adducts. This result is suggested to reflect greater ionic (CuII-O2-I) character within the valence bond description. A revised interpretation of the physical origins of the 18O EIEs upon O2 binding to redox metals is also advanced along with experimental data that should be used as benchmarks for interpreting 18O kinetic isotope effects upon enzyme reactions.

  18. Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin

    Science.gov (United States)

    Roche, Camille J.; Dantsker, David; Heller, Elizabeth R.; Sabat, Joseph E.; Friedman, Joel M.

    2013-08-01

    Hydration waters impact protein dynamics. Dissecting the interplay between hydration waters and dynamics requires a protein that manifests a broad range of dynamics. Proteins in reverse micelles (RMs) have promise as tools to achieve this objective because the water content can be manipulated. Hemoglobin is an appropriate tool with which to probe hydration effects. We describe both a protocol for hemoglobin encapsulation in reverse micelles and a facile method using PEG and cosolvents to manipulate water content. Hydration properties are probed using the water-sensitive fluorescence from Hb bound pyranine and covalently attached Badan. Protein dynamics are probed through ligand recombination traces derived from photodissociated carbonmonoxy hemoglobin on a log scale that exposes the potential role of both α and β solvent fluctuations in modulating protein dynamics. The results open the possibility of probing hydration level phenomena in this system using a combination of NMR and optical probes.

  19. Inference of protein diffusion probed via fluorescence correlation spectroscopy

    Science.gov (United States)

    Tsekouras, Konstantinos

    2015-03-01

    Fluctuations are an inherent part of single molecule or few particle biophysical data sets. Traditionally, ``noise'' fluctuations have been viewed as a nuisance, to be eliminated or minimized. Here we look on how statistical inference methods - that take explicit advantage of fluctuations - have allowed us to draw an unexpected picture of single molecule diffusional dynamics. Our focus is on the diffusion of proteins probed using fluorescence correlation spectroscopy (FCS). First, we discuss how - in collaboration with the Bustamante and Marqusee labs at UC Berkeley - we determined using FCS data that individual enzymes are perturbed by self-generated catalytic heat (Riedel et al, Nature, 2014). Using the tools of inference, we found how distributions of enzyme diffusion coefficients shift in the presence of substrate revealing that enzymes performing highly exothermic reactions dissipate heat by transiently accelerating their center of mass following a catalytic reaction. Next, when molecules diffuse in the cell nucleus they often appear to diffuse anomalously. We analyze FCS data - in collaboration with Rich Day at the IU Med School - to propose a simple model for transcription factor binding-unbinding in the nucleus to show that it may give rise to apparent anomalous diffusion. Here inference methods extract entire binding affinity distributions for the diffusing transcription factors, allowing us to precisely characterize their interactions with different components of the nuclear environment. From this analysis, we draw key mechanistic insight that goes beyond what is possible by simply fitting data to ``anomalous diffusion'' models.

  20. Preferred sites and pathways for electron transfer in blue copper proteins

    DEFF Research Database (Denmark)

    Farver, O; Pecht, I

    1988-01-01

    probably also coordinated to carboxylate groups, present in plastocyanin, and in stellacyanin 12 A and 6 A, respectively, from the copper center. The salient feature emerging from examination of the three copper proteins is that a pi-facilitated electron transfer (E.T.) pathway may be operative; in azurin......, E.T. proceeds via an extended imidazole ring system, and in plastocyanin and stellacyanin via a weakly coupled pi-system. Therefore, a case emerges for suggesting that this is the common feature of the long-distance intramolecular E.T. in this class of metalloproteins. These pathways are most...... probably a regulatory alternative to the E.T. site recognized at the exposed, "Northern" imidazole coordinated to copper in all these proteins....

  1. Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells.

    Science.gov (United States)

    Wan, Qingwen; Okashah, Najeah; Inoue, Asuka; Nehmé, Rony; Carpenter, Byron; Tate, Christopher G; Lambert, Nevin A

    2018-03-09

    G protein-coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefitted greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm, and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase-mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells, and should be useful for discovering and characterizing G protein subtype-biased ligands. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Probing the production of amidated peptides following genetic and dietary copper manipulations.

    Directory of Open Access Journals (Sweden)

    Ping Yin

    Full Text Available Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM, the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide is converted into a reaction intermediate (hydroxyglycine-extended peptide by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/- via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/- mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/- mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.

  3. Is copper(I) really soft? Probing the hardness of Cu(I) with ...

    Indian Academy of Sciences (India)

    Unknown

    Probing the hardness of Cu(I) with pyridinecarboxaldehyde ligands. D SARAVANABHARATHI, M NETHAJI and A G SAMUELSON*. Department of Inorganic and Physical Chemistry, Indian Institute of Science,. Bangalore 560 012, India e-mail: ashoka@ipc.iisc.ernet.in. Abstract. Cu(I) complexes of formula Cu(PPh3)2LClO4 ...

  4. Is copper(I) really soft? Probing the hardness of Cu(I) with ...

    Indian Academy of Sciences (India)

    Unknown

    (larger the pKa, stronger the donation) than that of 2-pyridinecarboxaldehyde are expected to compete with the chelated structure. A four-fold excess of a non chelating pyridine was used to probe the strength of the chelating ligand in complex 1. The binding of pyridine and their pKa values show a clear relationship (figure 6 ...

  5. The Methylococcus capsulatus (Bath secreted protein, MopE*, binds both reduced and oxidized copper.

    Directory of Open Access Journals (Sweden)

    Thomas Ve

    Full Text Available Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES, and Electron Paramagnetic Resonance (EPR studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II at two weaker binding sites. Both Cu(II binding sites have properties typical of non-blue type II Cu (II centres, and the strongest of the two Cu(II sites is characterised by a relative high hyperfine coupling of copper (A(|| =20 mT. Immobilized metal affinity chromatography binding studies suggests that residues in the N-terminal part of MopE* are involved in forming binding site(s for Cu(II ions. Our results support the hypothesis that MopE plays an important role in copper uptake, possibly making use of both its high (Cu(I and low Cu(II affinity properties.

  6. Relative and combined effects of ethanol and protein deficiency on bone manganese and copper.

    Science.gov (United States)

    González-Pérez, José M; González-Reimers, Emilio; DeLaVega-Prieto, María José; Durán-Castellón, María del Carmen; Viña-Rodríguez, José; Galindo-Martín, Luis; Alvisa-Negrín, Julio; Santolaria-Fernández, Francisco

    2012-06-01

    Both manganese and copper may affect bone synthesis. Bone content of both metals can be altered in alcoholics, although controversy exists regarding this matter. To analyse the relative and combined effects of ethanol and a low protein diet on bone copper and manganese, and their relationships with bone structure and metabolism, including trabecular bone mass (TBM), osteoid area (OA), osteocalcin (OCN), insulin-like growth factor-1 (IGF-1), parathyroid hormone (PTH), urinary hydroxyproline (uHP) and vitamin D. Adult male Sprague-Dawley rats were divided into four groups. The control rats received a 18% protein-containing diet; a second group, an isocaloric, 2% protein-containing diet; a third one, an isocaloric, 36% ethanol-containing diet and a fourth, an isocaloric diet containing 2% protein and 36% ethanol. After sacrifice, TBM and OA were histomorphometrically assessed; bone and serum manganese and copper were determined by atomic absorption spectrophotometry, and serum OCN, IGF-1, PTH, uHP and vitamin D by radioimmunoassay. Ethanol-fed rats showed decreased TBM and bone manganese. Significant relationships existed between bone manganese and TBM, serum IGF-1 and OCN. Ethanol leads to a decrease in bone manganese, related to decreased bone mass and bone synthesis. No alterations were found in bone copper.

  7. Photolytic Cross-Linking to Probe Protein-Protein and Protein-Matrix Interactions in Lyophilized Powders.

    Science.gov (United States)

    Iyer, Lavanya K; Moorthy, Balakrishnan S; Topp, Elizabeth M

    2015-09-08

    Protein structure and local environment in lyophilized formulations were probed using high-resolution solid-state photolytic cross-linking with mass spectrometric analysis (ssPC-MS). In order to characterize structure and microenvironment, protein-protein, protein-excipient, and protein-water interactions in lyophilized powders were identified. Myoglobin (Mb) was derivatized in solution with the heterobifunctional probe succinimidyl 4,4'-azipentanoate (SDA) and the structural integrity of the labeled protein (Mb-SDA) confirmed using CD spectroscopy and liquid chromatography/mass spectrometry (LC-MS). Mb-SDA was then formulated with and without excipients (raffinose, guanidine hydrochloride (Gdn HCl)) and lyophilized. The freeze-dried powder was irradiated with ultraviolet light at 365 nm for 30 min to produce cross-linked adducts that were analyzed at the intact protein level and after trypsin digestion. SDA-labeling produced Mb carrying up to five labels, as detected by LC-MS. Following lyophilization and irradiation, cross-linked peptide-peptide, peptide-water, and peptide-raffinose adducts were detected. The exposure of Mb side chains to the matrix was quantified based on the number of different peptide-peptide, peptide-water, and peptide-excipient adducts detected. In the absence of excipients, peptide-peptide adducts involving the CD, DE, and EF loops and helix H were common. In the raffinose formulation, peptide-peptide adducts were more distributed throughout the molecule. The Gdn HCl formulation showed more protein-protein and protein-water adducts than the other formulations, consistent with protein unfolding and increased matrix interactions. The results demonstrate that ssPC-MS can be used to distinguish excipient effects and characterize the local protein environment in lyophilized formulations with high resolution.

  8. A selectively rhodamine-based colorimetric probe for detecting copper(II) ion

    Science.gov (United States)

    Zhang, Jiangang; Zhang, Li; Wei, Yanli; Chao, Jianbing; Shuang, Shaomin; Cai, Zongwei; Dong, Chuan

    2014-11-01

    A novel rhodamine derivative 3-bromo-5-methylsalicylaldehyde rhodamine B hydrazone (BMSRH) has been synthesized by reacting rhodamine B hydrazide with 3-bromo-5-methylsalicylaldehyde and developed as a new colorimetric probe for the selective and sensitive detection of Cu2+. Addition of Cu2+ to the solution of BMSRH results in a rapid color change from colorless to red together with an obvious new band appeared at 552 nm in the UV-vis absorption spectra. This change is attributed to the spirocycle form of BMSRH opened via coordination with Cu2+ in a 1:1 stoichiometry and their association constant is determined as 3.2 × 104 L mol-1. Experimental results indicate that the BMSRH can provide a rapid, selective and sensitive response to Cu2+ with a linear dynamic range 0.667-240 μmol/L. Common interferent ions do not show any interference on the Cu2+ determination. It is anticipated that BMSRH can be a good candidate probe and has potential application for Cu2+ determination. The proposed probe exhibits the following advantages: a quick, simple and facile synthesis.

  9. Scanning Hall Probe Microscopy of Magnetic Vortices inVery Underdoped yttrium-barium-copper-oxide

    Energy Technology Data Exchange (ETDEWEB)

    Guikema, Janice Wynn; /SLAC, SSRL

    2005-12-02

    Since their discovery by Bednorz and Mueller (1986), high-temperature cuprate superconductors have been the subject of intense experimental research and theoretical work. Despite this large-scale effort, agreement on the mechanism of high-T{sub c} has not been reached. Many theories make their strongest predictions for underdoped superconductors with very low superfluid density n{sub s}/m*. For this dissertation I implemented a scanning Hall probe microscope and used it to study magnetic vortices in newly available single crystals of very underdoped YBa{sub 2}Cu{sub 3}O{sub 6+x} (Liang et al. 1998, 2002). These studies have disproved a promising theory of spin-charge separation, measured the apparent vortex size (an upper bound on the penetration depth {lambda}{sub ab}), and revealed an intriguing phenomenon of ''split'' vortices. Scanning Hall probe microscopy is a non-invasive and direct method for magnetic field imaging. It is one of the few techniques capable of submicron spatial resolution coupled with sub-{Phi}{sub 0} (flux quantum) sensitivity, and it operates over a wide temperature range. Chapter 2 introduces the variable temperature scanning microscope and discusses the scanning Hall probe set-up and scanner characterizations. Chapter 3 details my fabrication of submicron GaAs/AlGaAs Hall probes and discusses noise studies for a range of probe sizes, which suggest that sub-100 nm probes could be made without compromising flux sensitivity. The subsequent chapters detail scanning Hall probe (and SQUID) microscopy studies of very underdoped YBa{sub 2}Cu{sub 3}O{sub 6+x} crystals with T{sub c} {le} 15 K. Chapter 4 describes two experimental tests for visons, essential excitations of a spin-charge separation theory proposed by Senthil and Fisher (2000, 2001b). We searched for predicted hc/e vortices (Wynn et al. 2001) and a vortex memory effect (Bonn et al. 2001) with null results, placing upper bounds on the vison energy inconsistent with

  10. A selectively fluorescein-based colorimetric probe for detecting copper(II) ion

    Science.gov (United States)

    Zhang, Li; Zhang, Xianhong

    2014-12-01

    A novel fluorescein derivative 3-bromo-5-methylsalicylaldehyde fluorescein hydrazone (BMSFH) has been synthesized by reacting fluorescein hydrazide with 3-bromo-5-methylsalicylaldehyde and was developed as a new colorimetric probe for detection of Cu2+. In the presence of Cu2+ the BMSFH exhibits a rapid color change from colorless to yellow together with an obvious new band appeared at 502 nm in the UV-vis absorption spectra. However, other common alkali-, alkaline earth-, transition- and rare earth metal ions induced no or minimal spectral changes. This change is attributed to BMSFH via coordination with Cu2+ in a 1:1 stoichiometry and this binding to Cu2+ is reversible, as indicated by the bleaching of the color when the Cu2+ is extracted with EDTA. Experimental results indicate that the BMSFH can provide a rapid, selective and sensitive response to Cu2+ with a linear dynamic range 3.0-330 μmol/L and can be used as a potential Cu2+ colorimetric probe in aqueous solution.

  11. Overexpression of amyloid precursor protein increases copper content in HEK293 cells

    Energy Technology Data Exchange (ETDEWEB)

    Suazo, Miriam; Hodar, Christian; Morgan, Carlos [INTA, Laboratorio de Bioinformatica y Expresion Genica, Universidad de Chile, El Libano 5524, Macul, Santiago (Chile); Cerpa, Waldo [Centro de Envejecimiento y Regeneracion (CARE), Centro de Regulacion Celular y Patologia ' Joaquin V. Luco' (CRCP), MIFAB, Departamento de Biologia Celular y Molecular, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago (Chile); Cambiazo, Veronica [INTA, Laboratorio de Bioinformatica y Expresion Genica, Universidad de Chile, El Libano 5524, Macul, Santiago (Chile); Millenium Nucleus CGC, Universidad de Chile (Chile); Inestrosa, Nibaldo C. [Centro de Envejecimiento y Regeneracion (CARE), Centro de Regulacion Celular y Patologia ' Joaquin V. Luco' (CRCP), MIFAB, Departamento de Biologia Celular y Molecular, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago (Chile); Gonzalez, Mauricio, E-mail: mgonzale@inta.cl [INTA, Laboratorio de Bioinformatica y Expresion Genica, Universidad de Chile, El Libano 5524, Macul, Santiago (Chile)

    2009-05-15

    Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer's disease. However, its physiological function remains elusive. Cu{sup 2+} binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu{sup 2+} reduction and {sup 64}Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu{sup 2+} reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu{sup 2+} ions. Moreover, wild-type cells exposed to both Cu{sup 2+} ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu{sup 2+} reductase activity and increased {sup 64}Cu uptake. We conclude that Cu{sup 2+} reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.

  12. A sensitive fluorescent probe for the polar solvation dynamics at protein-surfactant interfaces.

    Science.gov (United States)

    Singh, Priya; Choudhury, Susobhan; Singha, Subhankar; Jun, Yongwoong; Chakraborty, Sandipan; Sengupta, Jhimli; Das, Ranjan; Ahn, Kyo-Han; Pal, Samir Kumar

    2017-05-17

    Relaxation dynamics at the surface of biologically important macromolecules is important taking into account their functionality in molecular recognition. Over the years it has been shown that the solvation dynamics of a fluorescent probe at biomolecular surfaces and interfaces account for the relaxation dynamics of polar residues and associated water molecules. However, the sensitivity of the dynamics depends largely on the localization and exposure of the probe. For noncovalent fluorescent probes, localization at the region of interest in addition to surface exposure is an added challenge compared to the covalently attached probes at the biological interfaces. Here we have used a synthesized donor-acceptor type dipolar fluorophore, 6-acetyl-(2-((4-hydroxycyclohexyl)(methyl)amino)naphthalene) (ACYMAN), for the investigation of the solvation dynamics of a model protein-surfactant interface. A significant structural rearrangement of a model histone protein (H1) upon interaction with anionic surfactant sodium dodecyl sulphate (SDS) as revealed from the circular dichroism (CD) studies is nicely corroborated in the solvation dynamics of the probe at the interface. The polarization gated fluorescence anisotropy of the probe compared to that at the SDS micellar surface clearly reveals the localization of the probe at the protein-surfactant interface. We have also compared the sensitivity of ACYMAN with other solvation probes including coumarin 500 (C500) and 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran (DCM). In comparison to ACYMAN, both C500 and DCM fail to probe the interfacial solvation dynamics of a model protein-surfactant interface. While C500 is found to be delocalized from the protein-surfactant interface, DCM becomes destabilized upon the formation of the interface (protein-surfactant complex). The timescales obtained from this novel probe have also been compared with other femtosecond resolved studies and molecular dynamics simulations.

  13. Detection of DNA hybridization by various spectroscopic methods using the copper tetraphenylporphyrin complex as a probe

    International Nuclear Information System (INIS)

    Gong, H.; Cai, C.; Ma, Y.; Chen, X.

    2012-01-01

    We are presenting new and highly sensitive hybridization assays. They are based on various spectroscopic methods including resonance light scattering, circular dichroism, ultraviolet spectra and fluorescence spectra, as well as atomic force microscopy, and relies on the interaction of the Cu(II), Ni(II), Mg(II), Co(II), Cd(II), and Zn(II) complexes, respectively, of tetraphenylporphyrin (TPP) with double-strand DNA (dsDNA) and single strand DNA (ssDNA). The interaction results in amplified resonance light scattering (RLS) signals and enables the detection of hybridization without the need for labeling DNA. The RLS signals are strongest in case of the Cu (II)-TPP complex which therefore was selected as the probe. The technique is simple, robust, accurate, and can be completed in less than one hour. (author)

  14. Comparative time-courses of copper-ion-mediated protein and lipid oxidation in low-density lipoprotein

    DEFF Research Database (Denmark)

    Knott, Heather M; Baoutina, Anna; Davies, Michael Jonathan

    2002-01-01

    -free BSA. Concomitant with Trp loss, the antioxidant alpha-tocopherol is consumed with subsequent extensive lipid peroxidation. Further changes to the protein, including the copper-ion-dependent 3.5-fold increase in 3,4-dihydroxyphenylalanine and the copper-ion-independent 3-5-fold increase in o...

  15. Copper and ectopic expression of the Arabidopsis transport protein COPT1 alter iron homeostasis in rice (Oryza sativa L.).

    Science.gov (United States)

    Andrés-Bordería, Amparo; Andrés, Fernando; Garcia-Molina, Antoni; Perea-García, Ana; Domingo, Concha; Puig, Sergi; Peñarrubia, Lola

    2017-09-01

    Copper deficiency and excess differentially affect iron homeostasis in rice and overexpression of the Arabidopsis high-affinity copper transporter COPT1 slightly increases endogenous iron concentration in rice grains. Higher plants have developed sophisticated mechanisms to efficiently acquire and use micronutrients such as copper and iron. However, the molecular mechanisms underlying the interaction between both metals remain poorly understood. In the present work, we study the effects produced on iron homeostasis by a wide range of copper concentrations in the growth media and by altered copper transport in Oryza sativa plants. Gene expression profiles in rice seedlings grown under copper excess show an altered expression of genes involved in iron homeostasis compared to standard control conditions. Thus, ferritin OsFER2 and ferredoxin OsFd1 mRNAs are down-regulated whereas the transcriptional iron regulator OsIRO2 and the nicotianamine synthase OsNAS2 mRNAs rise under copper excess. As expected, the expression of OsCOPT1, which encodes a high-affinity copper transport protein, as well as other copper-deficiency markers are down-regulated by copper. Furthermore, we show that Arabidopsis COPT1 overexpression (C1 OE ) in rice causes root shortening in high copper conditions and under iron deficiency. C1 OE rice plants modify the expression of the putative iron-sensing factors OsHRZ1 and OsHRZ2 and enhance the expression of OsIRO2 under copper excess, which suggests a role of copper transport in iron signaling. Importantly, the C1 OE rice plants grown on soil contain higher endogenous iron concentration than wild-type plants in both brown and white grains. Collectively, these results highlight the effects of rice copper status on iron homeostasis, which should be considered to obtain crops with optimized nutrient concentrations in edible parts.

  16. In vitro thermodynamic dissection of human copper transfer from chaperone to target protein.

    Science.gov (United States)

    Niemiec, Moritz S; Weise, Christoph F; Wittung-Stafshede, Pernilla

    2012-01-01

    Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu) transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4). Atox1 and WD4 have the same fold (ferredoxin-like fold) and Cu-binding site (two surface exposed cysteine residues) and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4). We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4) of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer.

  17. Involvement of protein kinase D in expression and trafficking of ATP7B (copper ATPase).

    Science.gov (United States)

    Pilankatta, Rajendra; Lewis, David; Inesi, Giuseppe

    2011-03-04

    ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673. The presence of PKD protein in the microsomal fraction was demonstrated by Western blotting. PKD is a serine/threonine kinase that associates with the trans-Golgi network, regulating fission of transport carriers destined to the cell surface. Parallel studies on cultured cells showed that nascent WT ATP7B transits to the Golgi complex where it undergoes serine phosphorylation by PKD. Misfolded ATP7B protein (especially if subjected to deletions) underwent proteasome-mediated degradation, which provides effective quality control. Inhibition of proteasome-mediated degradation with MG132 yielded additional, but nonfunctional protein. On the other hand, serine phosphorylation protected WT ATP7B from degradation. Protection was enhanced by PKD activation with phorbol esters and limited by PKD inhibition with CID75673. As a final step, phosphorylated ATP7B was transferred from the Golgi complex to cytosolic trafficking vesicles. Phosphorylation and trafficking were completely prevented by mutations of critical copper binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. ATP7B trafficking was markedly reduced by the Ser-478/481/1121/1453 to Ala mutation. We conclude that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking.

  18. Comparative study between probe focussed sonication and conventional stirring in the evaluation of cadmium and copper in plants

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, Sara; Fonseca, Luis P. [Technical University of Lisbon, Centro de Engenharia Quimica e Biologica, Instituto Superior Tecnico, Lisbon (Portugal); Capelo, Jose L. [University of Vigo at Ourense Campus, Analytical and Food Chemistry Department, Science Faculty, Ourense (Spain); Armas, Teresa; Vilhena, Fernanda; Goncalves, Maria L.S.; Mota, A.M. [Technical University of Lisbon, Centro de Quimica Estrutural, Instituto Superior Tecnico, Lisbon (Portugal); Pinto, Ana P. [University of Evora, Herdade Experimental da Mitra, ICAAM-Instituto de Ciencias Agrarias e Ambientais Mediterranicas, Evora (Portugal)

    2010-11-15

    Ultrasound (US)-assisted extraction has been widely used for metal ion extraction in plants due to its unique properties of decreased extraction time, minimal contamination, low reagent consumption and low cost. However, very few papers present a sound comparison between probe-focussed sonication and conventional stirring in the evaluation of metal ion extraction in plants. In this study, ultrasonic-assisted digestion has been evaluated and compared to magnetic stirring for total copper and cadmium determination by atomic absorption spectrometry in biological samples (plants, plankton and mussels). The same experimental conditions of sample amount and particle size, extractant solution and extraction time were applied for both ultrasound and magnetic stirring-assisted extraction methods in order to truly compare their effect on metal ion solubilisation. To gain further insight in this issue, dried and fresh plants were tested. The results obtained indicated that osmotic tension in cell walls, produced when dried and powdered samples were immersed in the extractant solution, had an important contribution to metal ion solubilisation, the enhancement due to US for the same purpose being negligible. (orig.)

  19. Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

    Directory of Open Access Journals (Sweden)

    Juan A. González-Vera

    2015-11-01

    Full Text Available Probing the dynamic activities of protein kinases in real-time in living cells constitutes a major challenge that requires specific and sensitive tools tailored to meet the particular demands associated with cellular imaging. The development of genetically-encoded and synthetic fluorescent biosensors has provided means of monitoring protein kinase activities in a non-invasive fashion in their native cellular environment with high spatial and temporal resolution. Here, we review existing technologies to probe different dynamic features of protein kinases and discuss limitations where new developments are required to implement more performant tools, in particular with respect to infrared and near-infrared fluorescent probes and strategies which enable improved signal-to-noise ratio and controlled activation of probes.

  20. Probing folding free energy landscape of small proteins through ...

    Indian Academy of Sciences (India)

    Unknown

    lattice and off-lattice models of proteins have been used to study the statistical and dynamical aspects of folding.12,13 Levitt pioneered in the computational studies of protein folding using off-lattice protein models.14 A recent off-lattice model study of HP-36 based on hydrophobicity tried to correlate the folding with many ...

  1. Development of a generic approach to native metalloproteomics: application to the quantitative identification of soluble copper proteins in Escherichia coli.

    Science.gov (United States)

    Sevcenco, Ana-Maria; Krijger, Gerard C; Pinkse, Martijn W H; Verhaert, Peter D E M; Hagen, Wilfred R; Hagedoorn, Peter-Leon

    2009-05-01

    A combination of techniques to separate and quantify the native proteins associated with a particular transition metal ion from a cellular system has been developed. The procedure involves four steps: (1) labeling of the target proteins with a suitable short-lived radioisotope (suitable isotopes are (64)Cu, (67)Cu, (187)W, (99)Mo, (69)Zn, (56)Mn, (65)Ni); (2) separation of intact soluble holoproteins using native isoelectric focusing combined with blue native polyacrylamide gel electrophoresis into native-native 2D gel electrophoresis; (3) spot visualization and quantification using autoradiography; and (4) protein identification with tandem mass spectrometry. The method was applied to the identification of copper proteins from a soluble protein extract of wild-type Escherichia coli K12 using the radioisotope (64)Cu. The E. coli protein CueO, which has previously been only identified as a multicopper oxidase following homologous overexpression, was now directly detected as a copper protein against a wild-type background at an expression level of 0.007% of total soluble protein. The retention of the radioisotope by the copper proteins throughout the separation process corroborates the method to be genuinely native. The procedure developed here can be applied to cells of any origin, and to any metal having suitable radioisotopes. The finding that the periplasmic protein CueO is the only major form of soluble protein bound copper in E. coli strengthens the view that the bacterial periplasm contains only a few periplasmic copper proteins, and that the cytosol is devoid of copper proteins.

  2. Copper transport.

    Science.gov (United States)

    Linder, M C; Wooten, L; Cerveza, P; Cotton, S; Shulze, R; Lomeli, N

    1998-05-01

    In adult humans, the net absorption of dietary copper is approximately 1 mg/d. Dietary copper joins some 4-5 mg of endogenous copper flowing into the gastrointestinal tract through various digestive juices. Most of this copper returns to the circulation and to the tissues (including liver) that formed them. Much lower amounts of copper flow into and out of other major parts of the body (including heart, skeletal muscle, and brain). Newly absorbed copper is transported to body tissues in two phases, borne primarily by plasma protein carriers (albumin, transcuprein, and ceruloplasmin). In the first phase, copper goes from the intestine to the liver and kidney; in the second phase, copper usually goes from the liver (and perhaps also the kidney) to other organs. Ceruloplasmin plays a role in this second phase. Alternatively, liver copper can also exit via the bile, and in a form that is less easily reabsorbed. Copper is also present in and transported by other body fluids, including those bathing the brain and central nervous system and surrounding the fetus in the amniotic sac. Ceruloplasmin is present in these fluids and may also be involved in copper transport there. The concentrations of copper and ceruloplasmin in milk vary with lactational stage. Parallel changes occur in ceruloplasmin messenger RNA expression in the mammary gland (as determined in pigs). Copper in milk ceruloplasmin appears to be particularly available for absorption, at least in rats.

  3. Probing intracellular motor protein activity using an inducible cargo trafficking assay

    NARCIS (Netherlands)

    L.C. Kapitein (Lukas); M.A. Schlager (Max); W.A. van der Zwan (Wouter); P. Wulf (Phebe); N. Keijzer (Nanda); C.C. Hoogenraad (Casper)

    2010-01-01

    textabstractAlthough purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living

  4. Benzoselendiazole-based responsive long-lifetime photoluminiscent probes for protein kinases

    DEFF Research Database (Denmark)

    Ekambaram, R; Enkvist, E; Manoharan, GB

    2014-01-01

    Benzoselenadiazole-containing inhibitors of protein kinases were constructed and their capability to emit phosphorescence in the kinase-bound state was established. Labelling of the inhibitors with a red fluorescent dye led to sensitive responsive photoluminescent probes for protein kinase CK2...

  5. Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids

    DEFF Research Database (Denmark)

    Nåbo, Lina J.; Madsen, Charlotte S.; Jensen, Knud J.

    2015-01-01

    Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid...... targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild...

  6. Resonance Raman spectroscopy of amicyanin, a blue copper protein from Paracoccus denitrificans

    International Nuclear Information System (INIS)

    Sharma, K.D.; Loehr, T.M.; Sanders-Loehr, J.; Husain, M.; Davidson, V.L.

    1988-01-01

    The copper binding site of amicyanin from Paracoccus denitrificans has been examined by resonance Raman spectroscopy. The pattern of vibrational modes is clearly similar to those of the blue copper proteins azurin and plastocyanin. Intense resonance-enhanced peaks are observed at 377, 392, and 430 cm-1 as well as weaker overtones and combination bands in the high frequency region. Most of the peaks below 500 cm-1 shift 0.5-1.5 cm-1 to lower energy when the protein is exposed to D 2 O. Based on the pattern of conserved amino acids, the axial type EPR spectrum, and the resonance Raman spectrum, it is proposed that the copper binding site in amicyanin contains a Cu(II) ion in a distorted trigonal planar geometry with one cysteine and two histidine ligands and an axial methionine ligand at a considerably longer distance. Furthermore, the presence of multiple intense Raman peaks in the 400 cm-1 region which are sensitive to deuterium substitution leads to the conclusion that the Cu-S stretch is coupled with internal ligand vibrational modes and that the sulfur of the cysteine ligand is likely to be hydrogen-bonded to the polypeptide backbone

  7. Application of lipid peroxidation and protein oxidation biomarkers for oxidative damage in mammalian cells. A comparison with two fluorescent probes.

    Science.gov (United States)

    Orhan, H; Gurer-Orhan, H; Vriese, E; Vermeulen, N P E; Meerman, J H N

    2006-09-01

    We recently developed two biomarker sets for oxidative damage: one for determination of lipid peroxidation (LPO) degradation products; acetaldehyde, propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal, malondialdehyde and acetone, by a gas chromatography-electron capture detection method, and the other for protein oxidation products such as o,o'-dityrosine, by an isotope dilution high performance liquid chromatography-tandem mass spectrometry method. In the present study, we explored the possibility to utilize these biomarkers for determining the oxidative damage in liver mammalian cells in vitro. Two different treatments were chosen for inducing oxidative stress in Chinese Hamster ovary cells: menadione and copper plus hydrogen peroxide (Cu2+/H2O2). Cells were incubated with the model compounds in the presence or absence of vitamin E and C, and cytotoxicity was evaluated by a nuclear-dye method. Results were compared to two fluorescent probes, H2DCF-DA and C11 -BODIPY581/591, which have been used for determining the formation of free radicals in the cells. From ten LPO degradation products, eight were increased significantly following incubation with menadione in cell lysate or incubation media. Menadione-induced oxidative stress was also confirmed by oxidation of fluorescent probes. However, no increased formation of protein oxidation products was observed. Vitamin E and C did not diminish the formation of LPO degradation products that were increased by menadione. Although Cu2+/H2O2 did not induce oxidation of fluorescent probes, it induced formation of six out of ten LPO degradation products. Vitamin E and C did not diminish the formation of LPO degradation products; vitamin C even substantially increased the formation of acetaldehyde and propanal, which is in line with its reported prooxidant action under certain conditions. Vitamin C also caused two-fold increase in Cu2+/H2O2-induced o,o'-dityrosine formation when applied simultaneously. In

  8. Probing RNA-protein networks: biochemistry meets genomics.

    Science.gov (United States)

    Campbell, Zachary T; Wickens, Marvin

    2015-03-01

    RNA-protein interactions are pervasive. The specificity of these interactions dictates which RNAs are controlled by what protein. Here we describe a class of revolutionary new methods that enable global views of RNA-binding specificity in vitro, for both single proteins and multiprotein complexes. These methods provide insight into central issues in RNA regulation in living cells, including understanding the balance between free and bound components, the basis for exclusion of binding sites, detection of binding events in the absence of discernible regulatory elements, and new approaches to targeting endogenous transcripts by design. Comparisons of in vitro and in vivo binding provide a foundation for comprehensive understanding of the biochemistry of protein-mediated RNA regulatory networks. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. In vitro study of proteins surface activity by tritium probe

    International Nuclear Information System (INIS)

    Chernysheva, M.G.; Badun, G.A.

    2010-01-01

    A new technique for in vitro studies of biomacromolecules interactions, their adsorption at aqueous/organic liquid interfaces and distribution in the bulk of liquid/liquid systems was developed. The method includes (1) tritium labeling of biomolecules by tritium thermal activation method and (2) scintillation phase step with organic phase, which can be concerned as a model of cellular membrane. Two globular proteins lysozyme and human serum albumin tested. We have determined the conditions of tritium labeling when labeled by-products can be easy separated by means of dialysis and size-exclusion chromatography. Scintillation phase experiments were conducted for three types of organic liquids. Thus, the influences of the nature of organic phase on proteins adsorption and its distribution in the bulk of aqueous/organic liquid system were determined. It was found that proteins possess high surface activity at aqueous/organic liquid interface. Furthermore, values of hydrophobicity of globular proteins were found by the experiment. (author)

  10. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding.

    Science.gov (United States)

    Tottey, Steve; Waldron, Kevin J; Firbank, Susan J; Reale, Brian; Bessant, Conrad; Sato, Katsuko; Cheek, Timothy R; Gray, Joe; Banfield, Mark J; Dennison, Christopher; Robinson, Nigel J

    2008-10-23

    Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).

  11. Homogeneous protein analysis by magnetic core-shell nanorod probes

    KAUST Repository

    Schrittwieser, Stefan

    2016-03-29

    Studying protein interactions is of vital importance both to fundamental biology research and to medical applications. Here, we report on the experimental proof of a universally applicable label-free homogeneous platform for rapid protein analysis. It is based on optically detecting changes in the rotational dynamics of magnetically agitated core-shell nanorods upon their specific interaction with proteins. By adjusting the excitation frequency, we are able to optimize the measurement signal for each analyte protein size. In addition, due to the locking of the optical signal to the magnetic excitation frequency, background signals are suppressed, thus allowing exclusive studies of processes at the nanoprobe surface only. We study target proteins (soluble domain of the human epidermal growth factor receptor 2 - sHER2) specifically binding to antibodies (trastuzumab) immobilized on the surface of our nanoprobes and demonstrate direct deduction of their respective sizes. Additionally, we examine the dependence of our measurement signal on the concentration of the analyte protein, and deduce a minimally detectable sHER2 concentration of 440 pM. For our homogeneous measurement platform, good dispersion stability of the applied nanoprobes under physiological conditions is of vital importance. To that end, we support our measurement data by theoretical modeling of the total particle-particle interaction energies. The successful implementation of our platform offers scope for applications in biomarker-based diagnostics as well as for answering basic biology questions.

  12. Probing protein sequences as sources for encrypted antimicrobial peptides.

    Science.gov (United States)

    Brand, Guilherme D; Magalhães, Mariana T Q; Tinoco, Maria L P; Aragão, Francisco J L; Nicoli, Jacques; Kelly, Sharon M; Cooper, Alan; Bloch, Carlos

    2012-01-01

    Starting from the premise that a wealth of potentially biologically active peptides may lurk within proteins, we describe here a methodology to identify putative antimicrobial peptides encrypted in protein sequences. Candidate peptides were identified using a new screening procedure based on physicochemical criteria to reveal matching peptides within protein databases. Fifteen such peptides, along with a range of natural antimicrobial peptides, were examined using DSC and CD to characterize their interaction with phospholipid membranes. Principal component analysis of DSC data shows that the investigated peptides group according to their effects on the main phase transition of phospholipid vesicles, and that these effects correlate both to antimicrobial activity and to the changes in peptide secondary structure. Consequently, we have been able to identify novel antimicrobial peptides from larger proteins not hitherto associated with such activity, mimicking endogenous and/or exogenous microorganism enzymatic processing of parent proteins to smaller bioactive molecules. A biotechnological application for this methodology is explored. Soybean (Glycine max) plants, transformed to include a putative antimicrobial protein fragment encoded in its own genome were tested for tolerance against Phakopsora pachyrhizi, the causative agent of the Asian soybean rust. This procedure may represent an inventive alternative to the transgenic technology, since the genetic material to be used belongs to the host organism and not to exogenous sources.

  13. Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin☆

    OpenAIRE

    Roche, Camille J.; Dantsker, David; Heller, Elizabeth R.; Sabat, Joseph E.; Friedman, Joel M.

    2013-01-01

    Hydration waters impact protein dynamics. Dissecting the interplay between hydration waters and dynamics requires a protein that manifests a broad range of dynamics. Proteins in reverse micelles (RMs) have promise as tools to achieve this objective because the water content can be manipulated. Hemoglobin is an appropriate tool with which to probe hydration effects. We describe both a protocol for hemoglobin encapsulation in reverse micelles and a facile method using PEG and cosolvents to mani...

  14. Design and application of natural product derived probes for activity based protein profiling

    OpenAIRE

    Battenberg, Oliver Alexander

    2015-01-01

    The identification of new antibacterial protein targets by activity based protein profiling (ABPP) is an important approach to face the increasing emergence of resistant bacteria. The scope of this work focuses on three new strategies for the labeling of antibacterial protein-targets with natural product derived ABPP-probes: A.) Evaluation of the intrinsic photo-reactivity of α-pyrones and pyrimidones for use as photo-crosslinkers. B.) Synthesis of a benzophenone-tag that combines photo-cross...

  15. A fluorogenic probe for imaging protein S-nitrosylation in live cells.

    Science.gov (United States)

    Shao, Shiyi; Chen, Bo; Cheng, Juan; Wang, Chengkun; Zhang, Yanli; Shao, Lingxiao; Hu, Yongzhou; Han, Yifeng; Han, Feng; Li, Xin

    2017-08-15

    S-nitrosylation is a posttranslational modification of protein cysteine residues leading to the formation of S-nitrosothiols and its detection is crucial to understanding of redox regulation and NO-based signaling. Prototypical detection methods for S-nitrosylation are always carried out ex situ. However, the reversible nature and the tendency of transnitrosylation highlight the necessity of its probing in intact live biological contexts. Herein we provide a fluorogenic chemical probe for the detection of S-nitrosylation in live endothelial cells. The probe is weakly emissive alone and becomes highly fluorescent only after undergoing a reaction with S-nitrosothiols in live cellular environments. This probe features high degrees of specificity and desirable sensitivity. Furthermore, it has been successfully applied to image the dynamic change of protein S-nitrosylation in live endothelial cells. The applicability of the probe in complex biological systems has been additionally verified by imaging a known target of S-nitrosylation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in live cells. Due to the versatility exemplified, this probe holds great promise for exploring the role of protein S-nitrosylation in the pathophysiological process of a variety of vascular diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Glycan masking of Plasmodium vivax Duffy Binding Protein for probing protein binding function and vaccine development.

    Directory of Open Access Journals (Sweden)

    Sowmya Sampath

    Full Text Available Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP and the Duffy Antigen Receptor for Chemokines (DARC and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

  17. Probing protein-lipid interactions by FRET between membrane fluorophores

    Science.gov (United States)

    Trusova, Valeriya M.; Gorbenko, Galyna P.; Deligeorgiev, Todor; Gadjev, Nikolai

    2016-09-01

    Förster resonance energy transfer (FRET) is a powerful fluorescence technique that has found numerous applications in medicine and biology. One area where FRET proved to be especially informative involves the intermolecular interactions in biological membranes. The present study was focused on developing and verifying a Monte-Carlo approach to analyzing the results of FRET between the membrane-bound fluorophores. This approach was employed to quantify FRET from benzanthrone dye ABM to squaraine dye SQ-1 in the model protein-lipid system containing a polycationic globular protein lysozyme and negatively charged lipid vesicles composed of phosphatidylcholine and phosphatidylglycerol. It was found that acceptor redistribution between the lipid bilayer and protein binding sites resulted in the decrease of FRET efficiency. Quantification of this effect in terms of the proposed methodology yielded both structural and binding parameters of lysozyme-lipid complexes.

  18. The lumenal loop M672-P707 of the Menkes protein (ATP7A) transfers copper to peptidylglycine monooxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Otoikhian, Adenike [Oregon Health & Sciences University; Barry, Amanda N. [Los Alamos National Laboratory; Mayfield, Mary [Oregon Health & Science University; Nilges, Mark [Illinois EPR Center; Huang, Yiping [Johns Hopkins University; Lutsenko, Svetlana [Johns Hopkins University; Blackburn, Ninian [Oregon Health & Science University

    2012-05-14

    Copper transfer to cuproproteins located in vesicular compartments of the secretory pathway depends on activity of the copper translocating ATPase (ATP7A or ATP7B) but the mechanism of transfer is largely unexplored. Copper-ATPase ATP7A is unique in having a sequence rich in histidine and methionine residues located on the lumenal side of the membrane. The corresponding fragment binds Cu(I) when expressed as a chimera with a scaffold protein, and mutations or deletions of His and/or Met residues in its sequence inhibit dephosphorylation of the ATPase, a catalytic step associated with copper release. Here we present evidence for a potential role of this lumenal region of ATP7A in copper transfer to cuproenzymes. Both Cu(II) and Cu(I) forms were investigated since the form in which copper is transferred to acceptor proteins is currently unknown. Analysis of Cu(II) using EPR demonstrated that at Cu:P ratios below 1:1, 15N-substituted protein had Cu(II) bound by 4 His residues, but this coordination changed as the Cu(II) to protein ratio increased towards 2:1. XAS confirmed this coordination via analysis of the intensity of outer-shell scattering from imidazole residues. The Cu(II) complexes could be reduced to their Cu(I) counterparts by ascorbate, but here again, as shown by EXAFS and XANES spectroscopy, the coordination was dependent on copper loading. At low copper Cu(I) was bound by a mixed ligand set of His + Met while at higher ratios His coordination predominated. The copper-loaded loop was able to transfer either Cu(II) or Cu(I) to peptidylglycine monooxygenase in the presence of chelating resin, generating catalytically active enzyme in a process that appeared to involve direct interaction between the two partners. The variation of coordination with copper loading suggests copper-dependent conformational change which in turn could act as a signal for regulating copper release by the ATPase pump.

  19. Probing the extent of randomness in protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Joseph Ivanic

    Full Text Available Protein-protein interaction (PPI networks are commonly explored for the identification of distinctive biological traits, such as pathways, modules, and functional motifs. In this respect, understanding the underlying network structure is vital to assess the significance of any discovered features. We recently demonstrated that PPI networks show degree-weighted behavior, whereby the probability of interaction between two proteins is generally proportional to the product of their numbers of interacting partners or degrees. It was surmised that degree-weighted behavior is a characteristic of randomness. We expand upon these findings by developing a random, degree-weighted, network model and show that eight PPI networks determined from single high-throughput (HT experiments have global and local properties that are consistent with this model. The apparent random connectivity in HT PPI networks is counter-intuitive with respect to their observed degree distributions; however, we resolve this discrepancy by introducing a non-network-based model for the evolution of protein degrees or "binding affinities." This mechanism is based on duplication and random mutation, for which the degree distribution converges to a steady state that is identical to one obtained by averaging over the eight HT PPI networks. The results imply that the degrees and connectivities incorporated in HT PPI networks are characteristic of unbiased interactions between proteins that have varying individual binding affinities. These findings corroborate the observation that curated and high-confidence PPI networks are distinct from HT PPI networks and not consistent with a random connectivity. These results provide an avenue to discern indiscriminate organizations in biological networks and suggest caution in the analysis of curated and high-confidence networks.

  20. Probing the extent of randomness in protein interaction networks.

    Science.gov (United States)

    Ivanic, Joseph; Wallqvist, Anders; Reifman, Jaques

    2008-07-11

    Protein-protein interaction (PPI) networks are commonly explored for the identification of distinctive biological traits, such as pathways, modules, and functional motifs. In this respect, understanding the underlying network structure is vital to assess the significance of any discovered features. We recently demonstrated that PPI networks show degree-weighted behavior, whereby the probability of interaction between two proteins is generally proportional to the product of their numbers of interacting partners or degrees. It was surmised that degree-weighted behavior is a characteristic of randomness. We expand upon these findings by developing a random, degree-weighted, network model and show that eight PPI networks determined from single high-throughput (HT) experiments have global and local properties that are consistent with this model. The apparent random connectivity in HT PPI networks is counter-intuitive with respect to their observed degree distributions; however, we resolve this discrepancy by introducing a non-network-based model for the evolution of protein degrees or "binding affinities." This mechanism is based on duplication and random mutation, for which the degree distribution converges to a steady state that is identical to one obtained by averaging over the eight HT PPI networks. The results imply that the degrees and connectivities incorporated in HT PPI networks are characteristic of unbiased interactions between proteins that have varying individual binding affinities. These findings corroborate the observation that curated and high-confidence PPI networks are distinct from HT PPI networks and not consistent with a random connectivity. These results provide an avenue to discern indiscriminate organizations in biological networks and suggest caution in the analysis of curated and high-confidence networks.

  1. Probing protein ensemble rigidity and hydrogen-deuterium exchange.

    Science.gov (United States)

    Sljoka, Adnan; Wilson, Derek

    2013-10-01

    Protein rigidity and flexibility can be analyzed accurately and efficiently using the program floppy inclusion and rigid substructure topography (FIRST). Previous studies using FIRST were designed to analyze the rigidity and flexibility of proteins using a single static (snapshot) structure. It is however well known that proteins can undergo spontaneous sub-molecular unfolding and refolding, or conformational dynamics, even under conditions that strongly favor a well-defined native structure. These (local) unfolding events result in a large number of conformers that differ from each other very slightly. In this context, proteins are better represented as a thermodynamic ensemble of 'native-like' structures, and not just as a single static low-energy structure. Working with this notion, we introduce a novel FIRST-based approach for predicting rigidity/flexibility of the protein ensemble by (i) averaging the hydrogen bonding strengths from the entire ensemble and (ii) by refining the mathematical model of hydrogen bonds. Furthermore, we combine our FIRST-ensemble rigidity predictions with the ensemble solvent accessibility data of the backbone amides and propose a novel computational method which uses both rigidity and solvent accessibility for predicting hydrogen-deuterium exchange (HDX). To validate our predictions, we report a novel site specific HDX experiment which characterizes the native structural ensemble of Acylphosphatase from hyperthermophile Sulfolobus solfataricus (Sso AcP). The sub-structural conformational dynamics that is observed by HDX data, is closely matched with the FIRST-ensemble rigidity predictions, which could not be attained using the traditional single 'snapshot' rigidity analysis. Moreover, the computational predictions of regions that are protected from HDX and those that undergo exchange are in very good agreement with the experimental HDX profile of Sso AcP.

  2. Probing protein ensemble rigidity and hydrogen-deuterium exchange

    Science.gov (United States)

    Sljoka, Adnan; Wilson, Derek

    2013-10-01

    Protein rigidity and flexibility can be analyzed accurately and efficiently using the program floppy inclusion and rigid substructure topography (FIRST). Previous studies using FIRST were designed to analyze the rigidity and flexibility of proteins using a single static (snapshot) structure. It is however well known that proteins can undergo spontaneous sub-molecular unfolding and refolding, or conformational dynamics, even under conditions that strongly favor a well-defined native structure. These (local) unfolding events result in a large number of conformers that differ from each other very slightly. In this context, proteins are better represented as a thermodynamic ensemble of ‘native-like’ structures, and not just as a single static low-energy structure. Working with this notion, we introduce a novel FIRST-based approach for predicting rigidity/flexibility of the protein ensemble by (i) averaging the hydrogen bonding strengths from the entire ensemble and (ii) by refining the mathematical model of hydrogen bonds. Furthermore, we combine our FIRST-ensemble rigidity predictions with the ensemble solvent accessibility data of the backbone amides and propose a novel computational method which uses both rigidity and solvent accessibility for predicting hydrogen-deuterium exchange (HDX). To validate our predictions, we report a novel site specific HDX experiment which characterizes the native structural ensemble of Acylphosphatase from hyperthermophile Sulfolobus solfataricus (Sso AcP). The sub-structural conformational dynamics that is observed by HDX data, is closely matched with the FIRST-ensemble rigidity predictions, which could not be attained using the traditional single ‘snapshot’ rigidity analysis. Moreover, the computational predictions of regions that are protected from HDX and those that undergo exchange are in very good agreement with the experimental HDX profile of Sso AcP.

  3. Determinants of the relative reduction potentials of type-1 copper sites in proteins.

    Science.gov (United States)

    Li, Hui; Webb, Simon P; Ivanic, Joseph; Jensen, Jan H

    2004-06-30

    The relative Cu(2+)/Cu(+) reduction potentials of six type-1 copper sites (cucumber stellacyanin, P. aeruginosa azurin, poplar plastocyanin, C. cinereus laccase, T. ferrooxidans rusticyanin, and human ceruloplasmin), which lie in a reduction potential range from 260 mV to over 1000 mV, have been studied by quantum mechanical calculations. The range and relative orderings of the reduction potentials are reproduced very well compared to experimental values. The study suggests that the main structural determinants of the relative reduction potentials of the blue copper sites are located within 6 A of the Cu atoms. Further analysis suggests that the reduction potential differences of type-1 copper sites are caused by axial ligand interactions, hydrogen bonding to the S(Cys), and protein constraint on the inner sphere ligand orientations. The low reduction potential of cucumber stellacyanin is due mainly to a glutamine ligand at the axial position, rather than a methionine or a hydrophobic residue as in the other proteins. A stronger interaction with a backbone carbonyl group is a prime contributor to the lower reduction potential of P. aeruginosa azurin as compared to poplar plastocyanin, whereas the reverse is true for C. cinereus laccase and T. ferrooxidans rusticyanin. The lack of an axial methonine ligand also contributes significantly to the increased reduction potentials of C. cinereus laccase and human ceruloplasmin. However, in the case of C. cinereus laccase, this increase is attenuated by the presence of only one amide NH hydrogen bond to the S(Cys) rather than two in the other proteins. In human ceruloplasmin the reduction potential is further increased by the structural distortion of the equatorial ligand orientation.

  4. Controlled Immobilization Strategies to Probe Short Hyaluronan-Protein Interactions

    Science.gov (United States)

    Minsky, Burcu Baykal; Antoni, Christiane H.; Boehm, Heike

    2016-01-01

    Well-controlled grafting of small hyaluronan oligosaccharides (sHA) enables novel approaches to investigate biological processes such as angiogenesis, immune reactions and cancer metastasis. We develop two strategies for covalent attachment of sHA, a fast high-density adsorption and a two-layer system that allows tuning the density and mode of immobilization. We monitored the sHA adlayer formation and subsequent macromolecular interactions by label-free quartz crystal microbalance with dissipation (QCM-D). The modified surfaces are inert to unspecific protein adsorption, and yet retain the specific binding capacity of sHA. Thus they are an ideal tool to study the interactions of hyaluronan-binding proteins and short hyaluronan molecules as demonstrated by the specific recognition of LYVE-1 and aggrecan. Both hyaladherins recognize sHA and the binding is independent to the presence of the reducing end. PMID:26883791

  5. Copper binding to the N-terminal metal-binding sites or the CPC motif is not essential for copper-induced trafficking of the human Wilson protein (ATP7B)

    Science.gov (United States)

    Cater, Michael A.; La fontaine, Sharon; Mercer, Julian F. B.

    2006-01-01

    The Wilson protein (ATP7B) is a copper-translocating P-type ATPase that mediates the excretion of excess copper from hep-atocytes into bile. Excess copper causes the protein to traffic from the TGN (trans-Golgi network) to subapical vesicles. Using site-directed mutagenesis, mutations known or predicted to abrogate catalytic activity (copper translocation) were introduced into ATP7B and the effect of these mutations on the intracellular traf-ficking of the protein was investigated. Mutation of the critical aspartic acid residue in the phosphorylation domain (DKTGTIT) blocked copper-induced redistribution of ATP7B from the TGN, whereas mutation of the phosphatase domain [TGE (Thr-Gly-Glu)] trapped ATP7B at cytosolic vesicular compartments. Our findings demonstrate that ATP7B trafficking is regulated with its copper-translocation cycle, with cytosolic vesicular localization associated with the acyl-phosphate intermediate. In addition, mut-ation of the six N-terminal metal-binding sites and/or the trans-membrane CPC (Cys-Pro-Cys) motif did not suppress the consti-tutive vesicular localization of the ATP7B phosphatase domain mutant. These results suggested that copper co-ordination by these sites is not essential for trafficking. Importantly, copper-chelation studies with these mutants clearly demonstrated a requirement for copper in ATP7B trafficking, suggesting the presence of an additional copper-binding site(s) within the protein. The results presented in this report significantly advance our understanding of the regulatory mechanism that links copper-translocation activity with copper-induced intracellular trafficking of ATP7B, which is central to hepatic and hence systemic copper homoeostasis. PMID:16939419

  6. Identification and characterization of a novel Cut family cDNA that encodes human copper transporter protein CutC

    International Nuclear Information System (INIS)

    Li Jixi; Ji Chaoneng; Chen Jinzhong; Yang Zhenxing; Wang Yijing; Fei, Xiangwei; Zheng Mei; Gu Xing; Wen Ge; Xie Yi; Mao Yumin

    2005-01-01

    Copper is an essential heavy metal trace element that plays important roles in cell physiology. The Cut family was associated with the copper homeostasis and involved in several important metabolisms, such as uptake, storage, delivery, and efflux of copper. In this study, a novel Cut family cDNA was isolated from the human fetal brain library, which encodes a 273 amino acid protein with a molecular mass of about 29.3 kDa and a calculated pI of 8.17. It was named hCutC (human copper transporter protein CutC). The ORF of hCutC gene was cloned into pQE30 vector and expressed in Escherichia coli M15. The secreted hCutC protein was purified to a homogenicity of 95% by using the Ni-NTA affinity chromatography. RT-PCR analysis showed that the hCutC gene expressed extensively in human tissues. Subcellular location analysis of hCutC-EGFP fusion protein revealed that hCutC was distributed to cytoplasm of COS-7 cells, and both cytoplasm and nucleus of AD293 cells. The results suggest that hCutC may be one shuttle protein and play important roles in intracellular copper trafficking

  7. Peptide tag/probe pairs based on the coordination chemistry for protein labeling.

    Science.gov (United States)

    Uchinomiya, Shohei; Ojida, Akio; Hamachi, Itaru

    2014-02-17

    Protein-labeling methods serve as essential tools for analyzing functions of proteins of interest under complicated biological conditions such as in live cells. These labeling methods are useful not only to fluorescently visualize proteins of interest in biological systems but also to conduct protein and cell analyses by harnessing the unique functions of molecular probes. Among the various labeling methods available, an appropriate binding pair consisting of a short peptide and a de novo designed small molecular probe has attracted attention because of its wide utility and versatility. Interestingly, most peptide tag/probe pairs exploit metal-ligand coordination interactions as the main binding force responsible for their association. Herein, we provide an overview of the recent progress of these coordination-chemistry-based protein-labeling methods and their applications for fluorescence imaging and functional analysis of cellular proteins, while highlighting our originally developed labeling methods. These successful examples clearly exemplify the utility and versatility of metal coordination chemistry in protein functional analysis.

  8. Scanning Probe Optical Tweezers: a new tool to study DNA-protein interactions

    NARCIS (Netherlands)

    Huisstede, J.H.G.

    2006-01-01

    The main goal of the work described in this thesis is to construct a microscope in which OT and scanning probe microscopy (SPM) are combined, to be able to localize proteins while simultaneously controlling the tension within the DNA molecule. This apparatus enables the study of the effect of

  9. Environmentally sensitive probes for monitoring protein-membrane interactions at nanomolar concentrations

    Czech Academy of Sciences Publication Activity Database

    Shvadchak, Volodymyr V.; Kucherak, Oleksandr; Afitska, Kseniia; Dziuba, D.; Yushchenko, Dmytro A.

    2017-01-01

    Roč. 1859, č. 5 (2017), s. 852-859 ISSN 0005-2736 Institutional support: RVO:61388963 Keywords : solvatochromic probes * fluorescence * protein-membrane interaction * affinity * binding stoichiometry * alpha-synuclein Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.498, year: 2016

  10. Probing protein flexibility reveals a mechanism for selective promiscuity.

    Science.gov (United States)

    Pabon, Nicolas A; Camacho, Carlos J

    2017-04-22

    Many eukaryotic regulatory proteins adopt distinct bound and unbound conformations, and use this structural flexibility to bind specifically to multiple partners. However, we lack an understanding of how an interface can select some ligands, but not others. Here, we present a molecular dynamics approach to identify and quantitatively evaluate the interactions responsible for this selective promiscuity. We apply this approach to the anticancer target PD-1 and its ligands PD-L1 and PD-L2. We discover that while unbound PD-1 exhibits a hard-to-drug hydrophilic interface, conserved specific triggers encoded in the cognate ligands activate a promiscuous binding pathway that reveals a flexible hydrophobic binding cavity. Specificity is then established by additional contacts that stabilize the PD-1 cavity into distinct bound-like modes. Collectively, our studies provide insight into the structural basis and evolution of multiple binding partners, and also suggest a biophysical approach to exploit innate binding pathways to drug seemingly undruggable targets.

  11. Probes of the Mitochondrial cAMP-dependent Protein Kinase

    Science.gov (United States)

    Shell, Jennifer R.; Lawrence, David S.

    2013-01-01

    The development of a fluorescent assay to detect activity of the mitochondrial cAMP-dependent protein kinase (PKA) is described. A peptide-based sensor was utilized to quantify the relative amount of PKA activity present in each compartment of the mitochondria (the outer membrane, the intermembrane space, and the matrix). In the process of validating this assay, we discovered that PKA activity is regulated by the protease calpain. Upon exposure of bovine heart mitochondria to digitonin, Ca2+, and a variety of electron transport chain inhibitors, the regulatory subunits of the PKA holoenzyme (R2C2) are digested, releasing active catalytic subunits. This proteolysis is attenuated by calpain inhibitor I (ALLN). PMID:23410952

  12. Determination of proteins by a reverse biuret method combined with the copper-bathocuproine chelate reaction.

    Science.gov (United States)

    Matsushita, M; Irino, T; Komoda, T; Sakagishi, Y

    1993-07-16

    A method of protein determination has been developed which combines the biuret reaction and the copper(I)-bathocuproine chelate reaction. Protein in the specimen forms a Cu(2+)-protein chelate complex (biuret reaction) during the first step. Excess Cu2+ is reduced to Cu+ by ascrobic acid, allowing the Cu+ to form a Cu(+)-bathocuproine chelate complex during the second step. The amount of Cu(+)-bathocuproine chelate complex formed is inversely proportional to the protein concentration. The sensitivity (epsilon = 1.4 x 10(6) 1.mol-1.cm-1 against human albumin) of this method was higher than that of the original Lowry (9.8 x 10(5)), pyrogallol red (1.0 x 10(6)) and commercially available Coomassie Brilliant Blue G.250 methods (6.7 x 10(5)). The color intensities of human gamma-globulin, human globulin (fractions IV-1 and IV-4), bovine albumin, egg albumin and horse gamma-globulin against human albumin (100%) ranged from 92 to 101%. The results obtained with the present method (y) correlated well with those determined by the biuret method (r = 0.998, y = 0.98 chi - 0.002, x = 1.31, y = 1.29 g/l) in 30 diluted sera. These results confirm that this assay is similar in sensitivity to the original Lowry method, is rapid and has similar reactivity to each of the various proteins in biological fluids.

  13. EPR of Cu(II) in sarcosine cadmium chloride: probe into dopant site - symmetry and copper-sarcosine interaction

    CERN Document Server

    Pathinettam-Padiyan, D; Murugesan, R

    2000-01-01

    The electron paramagnetic resonance spectra of Cu(II) doped sarcosine cadmium chloride single crystals have been investigated at room temperature. Experimental results reveal that the Cu(II) ion enters the lattice interstitially. The observed superhyperfine lines indicate the superposition of two sets of quintet structure with interaction of nitrogen atoms and the two isotopes of copper. The spin Hamiltonian parameters are evaluated by Schonland method and the electric field symmetry around the copper ion is rhombic. An admixture of d sub z sup 2 orbital with the d sub x sub sup 2 sub - sub y sub sup 2 ground state is observed. Evaluation of MO coefficients reveals that the in-plane interaction between copper and nitrogen is strong in this lattice.

  14. Comparative time-courses of copper-ion-mediated protein and lipid oxidation in low-density lipoprotein

    DEFF Research Database (Denmark)

    Knott, Heather M; Baoutina, Anna; Davies, Michael Jonathan

    2002-01-01

    -courses of lipid and protein oxidation during copper-ion-mediated oxidation of low-density lipoprotein. We show that there is an early, lipid-mediated loss of 40-50% of the Trp residues of the apoB100 protein. There is no comparable loss over an identical period during the copper-ion-mediated oxidation of lipid......Free radicals damage both lipids and proteins and evidence has accumulated for the presence of both oxidised lipids and proteins in aged tissue samples as well as those from a variety of pathologies including atherosclerosis, diabetes, and Parkinson's disease. Oxidation of the protein and lipid...... moieties of low-density lipoprotein is of particular interest due to its potential role in the unregulated uptake of lipids and cholesterol by macrophages; this may contribute to the initial stage of foam cell formation in atherosclerosis. In the study reported here, we examined the comparative time...

  15. Mycobacterium tuberculosis copper-regulated protein SocB is an intrinsically disordered protein that folds upon interaction with a synthetic phospholipid bilayer.

    Science.gov (United States)

    Nowicka, Urszula; Hoffman, Morgan; Randles, Leah; Shi, Xiaoshan; Khavrutskii, Lyuba; Stefanisko, Karen; Tarasova, Nadya I; Darwin, K Heran; Walters, Kylie J

    2016-02-01

    Multiple genes in Mycobacterium tuberculosis (Mtb) are regulated by copper including socAB (small orf induced by copper A and B), which is induced by copper and repressed by RicR (regulated in copper repressor). socA and socB encode hypothetical proteins of 61 and 54 amino acids, respectively. Here, we use biophysical and computational methods to evaluate the SocB structure. We find that SocB lacks evidence for secondary structure, with no thermal cooperative unfolding event, according to circular dichroism measurements. 2D NMR spectra similarly exhibit hallmarks of a disordered structural state, which is also supported by analyzing SocB diffusion. Altogether, these findings suggest that by itself SocB is intrinsically disordered. Interestingly, SocB interacts with a synthetic phospholipid bilayer and becomes helical, which suggests that it may be membrane-associated. © 2015 Wiley Periodicals, Inc.

  16. Prediction of Reduction Potentials of Copper Proteins with Continuum Electrostatics and Density Functional Theory.

    Science.gov (United States)

    Fowler, Nicholas J; Blanford, Christopher F; Warwicker, Jim; de Visser, Sam P

    2017-11-02

    Blue copper proteins, such as azurin, show dramatic changes in Cu 2+ /Cu + reduction potential upon mutation over the full physiological range. Hence, they have important functions in electron transfer and oxidation chemistry and have applications in industrial biotechnology. The details of what determines these reduction potential changes upon mutation are still unclear. Moreover, it has been difficult to model and predict the reduction potential of azurin mutants and currently no unique procedure or workflow pattern exists. Furthermore, high-level computational methods can be accurate but are too time consuming for practical use. In this work, a novel approach for calculating reduction potentials of azurin mutants is shown, based on a combination of continuum electrostatics, density functional theory and empirical hydrophobicity factors. Our method accurately reproduces experimental reduction potential changes of 30 mutants with respect to wildtype within experimental error and highlights the factors contributing to the reduction potential change. Finally, reduction potentials are predicted for a series of 124 new mutants that have not yet been investigated experimentally. Several mutants are identified that are located well over 10 Å from the copper center that change the reduction potential by more than 85 mV. The work shows that secondary coordination sphere mutations mostly lead to long-range electrostatic changes and hence can be modeled accurately with continuum electrostatics. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  17. Protein surface topology-probing by selective chemical modification and mass spectrometric peptide mapping.

    OpenAIRE

    Suckau, D; Mak, M; Przybylski, M

    1992-01-01

    Aminoacetylation of lysine residues and the modification of arginine by 1,2-cyclohexanedione to N7,N8-(dihydroxy-1,2-cyclohexylidene)arginine were used for probing the surface topology of hen-eggwhite lysozyme as a model protein. The molecular identification of lysine and arginine modification sites was provided by molecular weight determinations of modified and unmodified tryptic peptide mixtures (peptide mapping) using 252Cf plasma desorption mass spectrometry. At conditions of limited chem...

  18. [Copper level and metallothionein-like Cu-binding protein in cultured skin fibroblasts from patients with Menkes' disease and Wilson's disease].

    Science.gov (United States)

    Sato, M; Hayashi, A; Ito, H; Tojo, M; Arima, M

    1984-11-01

    Copper concentration, intracellular copper distribution, and inducibility of metallothionein-like metal-binding protein (MLP) by copper or cadmium addition to culture medium were compared among three types of skin fibroblasts derived from patients with Menkes' disease and Wilson's disease, both exhibiting genetic defects of copper metabolism, and from normal subjects (control). Skin fibroblasts were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics in 5% CO2 at 37 degrees C. Cells were harvested with rubber-policeman, washed twice with phosphate-buffered saline, pH 7.2, suspended in deionized water, and homogenized. The homogenate from each cell type was used to determine the concentration of copper by atomic absorption spectrophotometry employing graphite-rod atomizer after lyophilization, ashing in HNO3, and coprecipitation with zirconium. Intracellular copper concentration was elevated in Menkes' cells (420 ng Cu/mg of protein) and Wilson's cells (217 ng Cu/mg of protein) than in control cells (90.0 ng Cu/mg of protein), although one of four Wilson's strains showed normal copper level (70.5 ng Cu/mg of protein). Cytosol copper concentration was 5.8-fold higher in Menkes' cells but only 1.3-fold in Wilson's cells than in control cells, and cytosol copper accounted for only 35% of total intracellular copper in Wilson's cells as compared with 68% and 52% in Menkes' and control cells, respectively. These suggest that accumulated copper in each cell type is differently distributed within cells; in Menkes' cells exclusively into cytosol, but in Wilson's cells into particulates rather than cytosol. Elution profiles from Sephadex G-75 columns indicated that most of copper had bound to MLP in Menkes' cells, though no Cu-MLP was detectable in Wilson's or control cells under these experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Nano-bio-optomechanics: nanoaperture tweezers probe single nanoparticles, proteins, and their interactions

    Science.gov (United States)

    Gordon, Reuven

    2015-09-01

    Nanoparticles in the single digit nanometer range can be easily isolated and studied with low optical powers using nanoaperture tweezers. We have studied individual proteins and their interactions with small molecules, DNA and antibodies. Recently, using the fluctuations of the trapped object, we have pioneered a new way to "listen" to the vibrations of nanoparticles in the 100 GHz - 1 THz range; the approach is called extraordinary acoustic Raman (EAR). EAR gives unprecedented low frequency spectra of individual proteins in solution, allowing for identification and analysis, as well as probing their role in biological functions. We have also used EAR to study the elastic properties, shape and size of various individual nanoparticles.

  20. Effect of copper on the characterization of proteins in the Spiny lobster, Panulirus homarus homarus (Linnaeus,1758

    Directory of Open Access Journals (Sweden)

    Maharajan Athisuyambulingam

    2014-07-01

    Full Text Available Copper is most toxic metal in marine organisms. Characterization of protein occurring in the metabolically active tissues of muscle (MU, hepatopancreas (HP and gills (GL of the spiny lobster, Panulirus homarus homarus on exposure to two sub-lethal doses (9.55 and 19.1 µg/l of copper were studied for 28 days of exposure (DoE. The electrophoretic pattern of muscle, hepatopancreas and gill proteins revealed 12, 8 and 8 slow moving bands (control. The number of bands decreased to 8 and 7, 6 and 5, 6 and 4 after 7 days of exposure to 9.55 µg/l and 19.1 µg/l concentrations of copper, respectively. After 28 days, the protein bands decreased to 7 and 6, 5 and 4, 4 and 4 at 9.55 µg/l and 19.1 µg/l concentrations of copper, respectively. Present study to indicate that to avoid the Cupro-Nickel coil in lobster holding centers in chiller plants used for cooling of water was found to be responsible for the mortality of lobsters during live transportation.

  1. A thiol probe for measuring unfolded protein load and proteostasis in cells.

    Science.gov (United States)

    Chen, Moore Z; Moily, Nagaraj S; Bridgford, Jessica L; Wood, Rebecca J; Radwan, Mona; Smith, Trevor A; Song, Zhegang; Tang, Ben Zhong; Tilley, Leann; Xu, Xiaohong; Reid, Gavin E; Pouladi, Mahmoud A; Hong, Yuning; Hatters, Danny M

    2017-09-07

    When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum. TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease.Proteostasis is maintained through a number of molecular mechanisms, some of which function to protect the folded state of proteins. Here the authors demonstrate the use of TPE-MI in a fluorigenic dye assay for the quantitation of unfolded proteins that can be used to assess proteostasis on a cellular or proteome scale.

  2. Develop Infrared Structural Biology for Probing Structural Dynamics of Protein Functions

    Science.gov (United States)

    Xie, Aihua; Kang, Zhouyang; Causey, Oliver; Liu, Charle

    2015-03-01

    Protein functions are carried out through a series of structural transitions. Lack of knowledge on functionally important structural motions of proteins impedes our understanding of protein functions. Infrared structural biology is an emerging technology with powerful applications for protein structural dynamics. One key element of infrared structural biology is the development of vibrational structural marker (VSM) database library that translates infrared spectroscopic signals into specific structural information. We report the development of VSM for probing the type, geometry and strength of hydrogen bonding interactions of buried COO- side chains of Asp and Glu in proteins. Quantum theory based first principle computational studies combined with bioinformatic hydrogen bond analysis are employed in this study. We will discuss the applications of VSM in mechanistic studies of protein functions. Infrared structural biology is expected to emerge as a powerful technique for elucidating the functional mechanism of a broad range of proteins, including water soluble and membrane proteins. This work is supported by OCAST HR10-078 and NSF DBI1338097.

  3. Probing the selectivity and protein·protein interactions of a nonreducing fungal polyketide synthase using mechanism-based crosslinkers.

    Science.gov (United States)

    Bruegger, Joel; Haushalter, Robert W; Haushalter, Bob; Vagstad, Anna L; Vagstad, Anna; Shakya, Gaurav; Mih, Nathan; Townsend, Craig A; Burkart, Michael D; Tsai, Shiou-Chuan

    2013-09-19

    Protein·protein interactions, which often involve interactions among an acyl carrier protein (ACP) and ACP partner enzymes, are important for coordinating polyketide biosynthesis. However, the nature of such interactions is not well understood, especially in the fungal nonreducing polyketide synthases (NR-PKSs) that biosynthesize toxic and pharmaceutically important polyketides. Here, we employ mechanism-based crosslinkers to successfully probe ACP and ketosynthase (KS) domain interactions in NR-PKSs. We found that crosslinking efficiency is closely correlated with the strength of ACP·KS interactions and that KS demonstrates strong starter unit selectivity. We further identified positively charged surface residues by KS mutagenesis, which mediates key interactions with the negatively charged ACP surface. Such complementary/matching contact pairs can serve as "adapter surfaces" for future efforts to generate new polyketides using NR-PKSs. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. A novel copper(II) coordination at His186 in full-length murine prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Yasuko [Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Hiraoka, Wakako [Laboratory of Biophysics, School of Science and Technology, Meiji University, Kawasaki 214-8571 (Japan); Igarashi, Manabu; Ito, Kimihito [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020 (Japan); Shimoyama, Yuhei [Soft-Matter Physics Laboratory, Graduate School of Emergent Science, Muroran Institute of Technology, Muroran 050-8585 (Japan); Horiuchi, Motohiro [Laboratory of Prion Diseases, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Yamamori, Tohru; Yasui, Hironobu; Kuwabara, Mikinori [Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Inagaki, Fuyuhiko [Laboratory of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812 (Japan); Inanami, Osamu, E-mail: inanami@vetmed.hokudai.ac.jp [Laboratory of Radiation Biology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan)

    2010-04-09

    To explore Cu(II) ion coordination by His{sup 186} in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP{sup C}.

  5. Colloid-probe AFM studies of the interaction forces of proteins adsorbed on colloidal crystals.

    Science.gov (United States)

    Singh, Gurvinder; Bremmell, Kristen E; Griesser, Hans J; Kingshott, Peter

    2015-04-28

    In recent years, colloid-probe AFM has been used to measure the direct interaction forces between colloidal particles of different size or surface functionality in aqueous media, as one can study different forces in symmerical systems (i.e., sphere-sphere geometry). The present study investigates the interaction between protein coatings on colloid probes and hydrophilic surfaces decorated with hexagonally close packed single particle layers that are either uncoated or coated with proteins. Controlled solvent evaporation from aqueous suspensions of colloidal particles (coated with or without lysozyme and albumin) produces single layers of close-packed colloidal crystals over large areas on a solid support. The measurements have been carried out in an aqueous medium at different salt concentrations and pH values. The results show changes in the interaction forces as the surface charge of the unmodified or modified particles, and ionic strength or pH of the solution is altered. At high ionic strength or pH, electrostatic interactions are screened, and a strong repulsive force at short separation below 5 nm dominates, suggesting structural changes in the absorbed protein layer on the particles. We also study the force of adhesion, which decreases with an increment in the salt concentration, and the interaction between two different proteins indicating a repulsive interaction on approach and adhesion on retraction.

  6. Tracking the Path of Carbon from Plants to Rhizobiomes using Protein Stable Isotope Probing

    Science.gov (United States)

    Lipton, M. S.; Nicora, C.; Shaw, J. B.; Karaoz, U.; Nuccio, E. E.; Zhalnina, K.; Paša-Tolić, L.; Brodie, E.; Pett-Ridge, J.; Firestone, M.

    2016-12-01

    Plants and microorganisms have complex relationships that mediate terrestrial carbon fluxes. Understanding the functional roles of belowground biotic communities and their interactions with plant roots is critical to understanding the fate of plant carbon in soil. Currently many `omics-driven technologies reveal only a static snap shot of functionality in biological communities, but temporally-resolved tools are necessary describe biological processes that change and evolve over time. Stable isotope probing (SIP) experiments are temporal in nature, and can track the flow of carbon from the atmosphere through the plant and into the soil microbial community through space and time, and ultimately reveal both plant-microbe interactions and the fate of the carbon within the system. The combination of 'omics approaches with SIP allows us to selectively target and characterize metabolically active organisms in a community, and elucidate functional potential over time-scales relevant to biological processes. Protein stable isotope probing (Pro-SIP) offers a unique perspective into the interaction between plants and microbial communities, and potentially links taxonomic, functional and metabolic information. We developed a Pro-SIP approach amenable for use in soil systems using soils collected from an annual grassland in Northern CA amended with 13C labeled glucose. Using this approach we determined the overall proteomic profile and recently synthesized proteins within the plant-associated microbial community. The mass spectrometric analysis of the proteins extracted from the soil identified over 60,000 proteins that correspond to about 2200 protein functions. Of these functions, over 500 were found to contain a labeled peptide. Proteins related to glucose metabolism, carbohydrate storage, sugar transport and sulfur metabolism were found to be actively synthesized after glucose amendment. In systems where plants were grown with 13CO2 revealed synthesis of central metabolism

  7. The Copper Metabolism MURR1 domain protein 1 (COMMD1 modulates the aggregation of misfolded protein species in a client-specific manner.

    Directory of Open Access Journals (Sweden)

    Willianne I M Vonk

    Full Text Available The Copper Metabolism MURR1 domain protein 1 (COMMD1 is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α. Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1 and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS, cause misfolding and aggregation of the mutant SOD1 (mSOD1 protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson's disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington's disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.

  8. Red-emitting BSA-stabilized copper nanoclusters acted as a sensitive probe for fluorescence sensing and visual imaging detection of rutin.

    Science.gov (United States)

    Wang, Benqian; Gui, Rijun; Jin, Hui; He, Weijie; Wang, Zonghua

    2018-02-01

    In this article, red-emitting copper nanoclusters (Cu NCs) were prepared in the matrix of bovine serum albumin (BSA) through using N 2 H 4 ·H 2 O as a reducing agent and BSA as a stabilizer. In the aqueous suspension of BSA-Cu NCs, the addition of rutin enabled the combination of rutin with BSA around Cu NCs via hydrogen bond and electrostatic interactions between functional groups of BSA and rutin. The approaching of rutin to Cu NCs induced that the fluorescence (FL) of Cu NCs was quenched by rutin due to the electron transfer from Cu NCs to rutin. Under the optimal conditions, the FL of Cu NCs was linearly decreased with the increase of rutin concentration from 0.1 to100 μM, together with a low detection limit of ca. 0.02μM. The FL quenching responses of Cu NCs were highly selective and sensitive towards rutin, over potential interferents in real human serum samples. Based on BSA-Cu NCs, this as-proposed FL probe exhibited a superior capability of rutin detection in real human serum samples. Significantly, this probe of rutin could realize the FL sensing of rutin and the naked-eye visual FL imaging detection of rutin in aqueous solution and on filter paper. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Investigation of the Copper Binding Site And the Role of Histidine As a Ligand in Riboflavin Binding Protein

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S.R.; Bencze, K.Z.; Russ, K.A.; Wasiukanis, K.; Benore-Parsons, M.; Stemmler, T.L.

    2009-05-26

    Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 {angstrom}, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a Cu-O{sub 3}N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit.

  10. The effect of occupational lead exposure on blood levels of zinc, iron, copper, selenium and related proteins.

    Science.gov (United States)

    Kasperczyk, Aleksandra; Prokopowicz, Adam; Dobrakowski, Michał; Pawlas, Natalia; Kasperczyk, Sławomir

    2012-12-01

    The study objective was to evaluate the effect of occupational lead exposure on blood concentrations of zinc, iron, copper, selenium and proteins related to them, such as transferrin, caeruloplasmin and haptoglobin. The examined group consisted of 192 healthy male employees of zinc-lead works. By the degree of lead exposure, the exposed group was subdivided into three subgroups. The control group was composed of 73 healthy male administrative workers. The markers of lead exposure (blood levels of lead and zinc protoporphyrin) were significantly elevated in the exposed group compared with the control group. Additionally, concentrations of copper and caeruloplasmin were raised. The significant increase in haptoglobin level was observed only in the low exposure group. Selenium levels were significantly decreased, whereas iron, zinc and transferrin levels were unchanged in the exposed group compared with the control group. There were positive correlations between the lead toxicity parameters and the copper and caeruloplasmin levels. In conclusion, the effect of occupational exposure to lead on the metabolism of trace metals appears to be limited. However, significant associations between lead exposure and levels of copper and selenium were shown. Changed levels of positive acute-phase proteins, such as caeruloplasmin and haptoglobin, were also observed.

  11. Nitrate as a probe of cytochrome c surface: crystallographic identification of crucial "hot spots" for protein-protein recognition.

    Science.gov (United States)

    De March, Matteo; Demitri, Nicola; De Zorzi, Rita; Casini, Angela; Gabbiani, Chiara; Guerri, Annalisa; Messori, Luigi; Geremia, Silvano

    2014-06-01

    The electrostatic surface of cytochrome c and its changes with the iron oxidation state are involved in the docking and undocking processes of this protein to its biological partners in the mitochondrial respiratory pathway. To investigate the subtle mechanisms of formation of productive macromolecular complexes and of their breakage following the electron transfer process, the X-ray structures of horse heart ferri-cytochrome c (trigonal form) and ferro-cytochrome c (monoclinic form) were obtained using nitrate ions both as a crystallizing agent and an anionic probe for mapping the electrostatic surface changes. Both crystal forms contain three protein molecules in the asymmetric unit. In addition, a total of 21.5 and 18 crystallographically independent nitrate ions were identified for the trigonal and monoclinic forms, respectively. By matching all the six crystallographically independent protein molecules, 26 different anion-protein interaction sites were identified on the surfaces of cytochrome c, 10 of which were found in both forms, 8 present only in the oxidized and 8 only in the reduced form. The structural analysis of the electron transfer complexes, based on this new information, suggests a specific exit strategy for cytochrome c after formation of productive protein-protein complexes: a directional sliding mechanism for the electron shuttle on the surface of the redox partner is proposed to take place after the electron transfer process has occurred. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Copper-mediated oxidative degradation of catecholamines and oxidative damage of protein

    Energy Technology Data Exchange (ETDEWEB)

    Goncalves, P.R.; Harria, M.I.N.; Felix, J.M.; Hoffmann, M.E. [Universidade Estadual de Campinas, SP (Brazil). Inst. de Biologia

    1997-12-31

    Full text. Degradative oxidation of catecholamines has been a matter of large interest in recent years due to the evidences associating their autoxidation with the etiology of neurotoxic and cardiotoxic processes. In this work we present data on the degradative oxidation of catecholamines of physiological importance: isoproterenol (IP), epinephrine (EP), norepinephrine (NEP), deoxyepinephrine (DEP) and dopamine (DA). The degradative oxidation of the catecholamines was followed by measurement of spectral changes and oxygen consumption by neutral aqueous solutions. The data show that Cu{sup 2+} strongly accelerated the rate of catecholamine oxidation, following the decreasing order; EP>DEP>IP>NEP>DA. The production of superoxide anion radical during catecholamine oxidation was very slow, even in the presence of Cu{sup 2+}. The ability of IP to induce damages on bovine serum albumin (BSA) was determined by measuring the formation of carbonyl-groups in the protein, detected by reduction with tritiated Na BH{sub 4}. The incubation of BSA with IP (50-500{mu}M), in the presence of 100{mu}M Cu{sup 2+} leaded to an increased and dose dependent {sup 3} H-incorporation by the oxidized protein. The production of oxidative damage by IP/Cu{sup 2+} was accompanied by marked BSA fragmentation, detected by SDS-polyacrylamide gel dependent (25-400{mu}M IP) des appearance of the original BSA band and appearance of smaller fragments spread in the gel, when incubation has been done in the presence of 100{mu}M Cu{sup 2+}. These results suggest that copper-catalysed oxidative degradation of proteins induced by catecholamines might be critically involved in the toxic action of these molecules

  13. Novel insights in the molecular pathogenesis of human copper homeostasis disorders through studies of protein-protein interactions

    NARCIS (Netherlands)

    Bie, P. de

    2007-01-01

    Copper is an essential element for living organisms, yet it is very toxic when present in amounts exceeding cellular needs. Delicate mechanisms have evolved to ensure proper copper homeostasis is maintained for the organism, as well as at a cellular level, and perturbations in these mechanisms give

  14. Proteolytic Scanning Calorimetry: A Novel Methodology that Probes the Fundamental Features of Protein Kinetic Stability

    Science.gov (United States)

    Tur-Arlandis, Gema; Rodriguez-Larrea, David; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2010-01-01

    We introduce proteolytic scanning calorimetry, a modification of the differential scanning calorimetry approach to the determination of protein stability in which a proteolytic enzyme (thermolysin) is used to mimic a harsh environment. This methodology allows the straightforward calculation of the rate of irreversible denaturation as a function of temperature and concentration of proteolytic enzyme and, as a result, has the potential to probe efficiently the fundamental biophysical features of protein kinetic stability. In the particular case of Escherichia coli thioredoxin (used as an illustrative example in this article), we find that the rate of irreversible denaturation is determined by 1), the global unfolding mechanism at low thermolysin concentrations, indicating that thermodynamic stability may contribute directly to the kinetic stability of thioredoxin under moderately harsh conditions and 2), the rate of unfolding at high thermolysin concentrations, indicating that the free-energy barrier for unfolding may act as a safety mechanism that ensures significant kinetic stability, even in very harsh environments. This thioredoxin picture, however, is by no means expected to be general and different proteins may show different patterns of kinetic stabilization. Proteolytic scanning calorimetry is particularly well-suited to probe this diversity at a fundamental biophysical level. PMID:20303845

  15. Fluorescence study of protein-lipid complexes with a new symmetric squarylium probe.

    Science.gov (United States)

    Ioffe, Valeriya M; Gorbenko, Galyna P; Deligeorgiev, Todor; Gadjev, Nikolai; Vasilev, Aleksey

    2007-06-01

    The novel symmetric squarylium derivative SQ-1 has been synthesized and tested for its sensitivity to the formation of protein-lipid complexes. SQ-1 binding to the model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in different molar ratios was found to be controlled mainly by hydrophobic interactions. Lysozyme (Lz) and ribonuclease A (RNase) exerted an influence on the probe association with lipid vesicles resulting presumably from the competition between SQ-1 and the proteins for bilayer free volume and modification of its properties. The magnitude of this effect was much higher for lysozyme which may stem from the amphipathy of protein alpha-helix involved in the membrane binding. Varying membrane composition provides evidence for the dye sensitivity to both hydrophobic and electrostatic protein-lipid interactions. Fluorescence anisotropy studies uncovered the restriction of SQ-1 rotational mobility in lipid environment in the presence of Lz and RNase being indicative of the incorporation of the proteins into bilayer interior. The results of binding, fluorescence quenching and kinetic experiments suggested lysozyme-induced local lipid demixing upon protein association with negatively charged membranes with threshold concentration of CL for the lipid demixing being 10 mol%.

  16. Transcriptomics as a tool to dissect copper homeostasis and COMMD protein function

    NARCIS (Netherlands)

    Müller, P.A.J.

    2008-01-01

    Gene expression profiling offers the possibility to objectively screen the complete genome for variations in gene expression in many different organisms in various defined environments. In the present work we aimed to gain novel insights in copper metabolism and in the function of COMMD (copper

  17. Development of a radioiodinated triazolopyrimidine probe for nuclear medical imaging of fatty acid binding protein 4.

    Directory of Open Access Journals (Sweden)

    Kantaro Nishigori

    Full Text Available Fatty acid binding protein 4 (FABP4 is the most well-characterized FABP isoform. FABP4 regulates inflammatory pathways in adipocytes and macrophages and is involved in both inflammatory diseases and tumor formation. FABP4 expression was recently reported for glioblastoma, where it may participate in disease malignancy. While FABP4 is a potential molecular imaging target, with the exception of a tritium labeled probe there are no reports of other nuclear imaging probes that target this protein. Here we designed and synthesized a nuclear imaging probe, [123I]TAP1, and evaluated its potential as a FABP4 targeting probe in in vitro and in vivo assays. We focused on the unique structure of a triazolopyrimidine scaffold that lacks a carboxylic acid to design the TAP1 probe that can undergo facilitated delivery across cell membranes. The affinity of synthesized TAP1 was measured using FABP4 and 8-anilino-1-naphthalene sulfonic acid. [125I]TAP1 was synthesized by iododestannylation of a precursor, followed by affinity and selectivity measurements using immobilized FABPs. Biodistributions in normal and C6 glioblastoma-bearing mice were evaluated, and excised tumors were subjected to autoradiography and immunohistochemistry. TAP1 and [125I]TAP1 showed high affinity for FABP4 (Ki = 44.5±9.8 nM, Kd = 69.1±12.3 nM. The FABP4 binding affinity of [125I]TAP1 was 11.5- and 35.5-fold higher than for FABP3 and FABP5, respectively. In an in vivo study [125I]TAP1 displayed high stability against deiodination and degradation, and moderate radioactivity accumulation in C6 tumors (1.37±0.24% dose/g 3 hr after injection. The radioactivity distribution profile in tumors partially corresponded to the FABP4 positive area and was also affected by perfusion. The results indicate that [125I]TAP1 could detect FABP4 in vitro and partly in vivo. As such, [125I]TAP1 is a promising lead compound for further refinement for use in in vivo FABP4 imaging.

  18. Probing protein: DNA interactions using a uniform monolayer of DNA and surface plasmon resonance

    Science.gov (United States)

    Shumaker-Parry, Jennifer S.; Campbell, Charles T.; Stormo, Gary D.; Silbaq, Fauzi S.; Aebersold, Rudolf H.

    2000-04-01

    A method is described for immobilizing double-stranded DNAs to a planar gold surface with high density and uniform spacing. This is accomplished by adsorbing biotinylated DNAs onto a nearly close-packed monolayer of the protein streptavidin. This streptavidin monolayer, which offers approximately 5 X 1012 biotin sites per cm2, is prepared first by adsorbing it onto a mixed self-assembled monolayer on gold which contains biotin-terminated and oligo-terminated alkylthiolates in a 3/7 ratio. This DNA- functionalized surface resists non-specific protein adsorption and is useful for probing the kinetics and equilibrium binding of proteins to DNA with surface plasmon resonance. This is demonstrated with the Mnt protein, which is found to bind in 3.8:1 ratio to its immobilized DNA operator sequence. This is consistent with its behavior in homogeneous solution, where it binds as a tetramer to its DNA. A sequence with a single base-pair mutation shows nearly as much Mnt binding, but a completely random DNA sequence shows only 5 percent of this binding. This proves that DNA-binding proteins bind sequence-specifically to double-stranded DNAs which are immobilized to gold with this streptavidin linker layer.

  19. Probing how initial retinal configuration controls photochemical dynamics in retinal proteins

    Directory of Open Access Journals (Sweden)

    Sheves M.

    2013-03-01

    Full Text Available The effects of the initial retinal configuration and the active isomerization coordinate on the photochemistry of retinal proteins (RPs are assessed by comparing photochemical dynamics of two stable retinal ground state configurations (all-trans,15-anti vs. 13-cis,15-syn, within two RPs: Bacteriorhodopsin (BR and Anabaena Sensory Rhodopsin (ASR. Hyperspectral pump-probe spectroscopy shows that photochemistry starting from 13-cis retinal in both proteins is 3-10 times faster than when started in the all-trans state, suggesting that the hastening is ubiquitous to microbial RPs, regardless of their different biological functions and origin. This may also relate to the known disparity of photochemical rates between microbial RPs and visual pigments. Importance and possible underlying mechanisms are discussed as well.

  20. A new carboxyl-copper-organic framework and its excellent selective absorbability for proteins

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Linyan [Department of Chemistry, Nankai University, Tianjin 300071 (China); Xin, Liangliang [School of Science, Tianjin University, Tianjin 300072 (China); Gu, Wen; Tian, Jinlei [Department of Chemistry, Nankai University, Tianjin 300071 (China); Tianjin Key Laboratory of Metal and Molecule Based Material Chemistry, Tianjin 300071 (China); Liao, Shengyun; Du, Peiyao; Tong, Yuzhang; Zhang, Yanping; Lv, Rui; Wang, Jingyao [Department of Chemistry, Nankai University, Tianjin 300071 (China); Liu, Xin, E-mail: liuxin64@nankai.edu.cn [Department of Chemistry, Nankai University, Tianjin 300071 (China); Tianjin Key Laboratory of Metal and Molecule Based Material Chemistry, Tianjin 300071 (China)

    2014-10-15

    .4. At the same time, XPS spectra were also investigated to verify the results. - Highlights: • A rare carboxyl-copper-organic framework has been hydrothermally synthesized. • An xfe-4-Fddd, (4{sup 2}.6.8{sup 3}) topology in 1 is created. • BSA was significantly less adsorbed onto the carboxylic acid-rich surface than lysozyme. • XPS spectra were also investigated to verify the results of protein adsorption.

  1. Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

    Science.gov (United States)

    2015-01-01

    Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental

  2. Probing the interaction of brain fatty acid binding protein (B-FABP with model membranes.

    Directory of Open Access Journals (Sweden)

    Fábio Dyszy

    Full Text Available Brain fatty acid-binding protein (B-FABP interacts with biological membranes and delivers polyunsaturated fatty acids (FAs via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called "portal region", formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that B-FABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs.

  3. Ester Carbonyl Vibration as a Sensitive Probe of Protein Local Electric Field

    Science.gov (United States)

    Pazos, Ileana M.; Ghosh, Ayanjeet; Tucker, Matthew J.; Gai, Feng

    2014-01-01

    The ability to quantify the local electrostatic environment of proteins and protein/peptide assemblies is key to yielding a microscopic understanding of many biological interactions and processes. Herein, we show that the ester carbonyl stretching vibration of two non-natural amino acids, L-aspartic acid 4-methyl ester and L-glutamic acid 5-methyl ester, is a convenient and sensitive probe in this regard since its frequency correlates linearly with the local electrostatic field for both hydrogen-bonding and non-hydrogen-bonding environments. We expect that the resultant frequency-electric field map will find use in various applications. In addition, we show that, when situated in a non-hydrogen bonding environment, this probe can also be used to measure the local dielectric constant (ε). For example, applying it to amyloid fibrils formed by Aβ16-22 reveals that the interior of such β-sheet assemblies has a ε of ~5.6. PMID:24788907

  4. Ester carbonyl vibration as a sensitive probe of protein local electric field.

    Science.gov (United States)

    Pazos, Ileana M; Ghosh, Ayanjeet; Tucker, Matthew J; Gai, Feng

    2014-06-10

    The ability to quantify the local electrostatic environment of proteins and protein/peptide assemblies is key to gaining a microscopic understanding of many biological interactions and processes. Herein, we show that the ester carbonyl stretching vibration of two non-natural amino acids, L-aspartic acid 4-methyl ester and L-glutamic acid 5-methyl ester, is a convenient and sensitive probe in this regard, since its frequency correlates linearly with the local electrostatic field for both hydrogen-bonding and non-hydrogen-bonding environments. We expect that the resultant frequency-electric-field map will find use in various applications. Furthermore, we show that, when situated in a non-hydrogen-bonding environment, this probe can also be used to measure the local dielectric constant (ε). For example, its application to amyloid fibrils formed by Aβ(16-22) revealed that the interior of such β-sheet assemblies has an ε value of approximately 5.6. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. A simple fluorescent probe for the fast sequential detection of copper and biothiols based on a benzothiazole derivative

    Science.gov (United States)

    Shen, Youming; Zhang, Xiangyang; Zhang, Chunxiang; Zhang, Youyu; Jin, Junling; Li, Haitao

    2018-02-01

    A simple benzothiazole fluorescent chemosensor was developed for the fast sequential detection of Cu2 + and biothiols through modulating the excited-state intramolecular proton transfer (ESIPT) process. The compound 1 exhibits highly selective and sensitive fluorescence ;on-off; recognition to Cu2 + with a 1:1 binding stoichiometry by ESIPT hinder. The in situ generated 1-Cu2 + complex can serve as an ;on-off; fluorescent probe for high selectivity toward biothiols via Cu2 + displacement approach, which exerts ESIPT recovery. It is worth pointing out that the 1-Cu2 + complex shows faster for cysteins (within 1 min) than other biothiols such as homocysteine (25 min) and glutathione (25 min). Moreover, the compound 1 displays 160 nm Stoke-shift for reversibly monitoring Cu2 + and biothiols. In addition, the probe is successfully used for fluorescent cellular imaging. This strategy via modulation the ESIPT state has been used for determination of Cu2 + and Cys with satisfactory results, which further demonstrates its value of practical applications.

  6. Spectrophotometric total protein assay with copper(II)-neocuproine reagent in alkaline medium.

    Science.gov (United States)

    Sözgen, Kevser; Cekic, Sema Demirci; Tütem, Esma; Apak, Resat

    2006-02-28

    Total protein assay was made using copper(II)-neocuproine (Nc) reagent in alkaline medium (with the help of a hydroxide-carbonate-tartarate solution) after 30min incubation at 40 degrees C. The absorbance of the reduction product, Cu(I)-Nc complex, was recorded at 450nm against a reagent blank. The absorptivity of the developed method for bovine serum albumin (BSA) was 0.023lmg(-1)cm(-1), greater than that of Lowry assay (0.0098), and much greater than that of Cu(II)-bicinchoninic acid (BCA) assay (0.00077). The linear range of the developed method (8-100mgl(-1) BSA) was as wide as that of Lowry, and much wider than that of BCA (200-1000mgl(-1) BSA) assay. The sensitivity of the method was greater than those of Cu-based assays (biuret, Lowry, and BCA) with a LOD of 1mgl(-1) BSA. The within-run and between-run precisions as RSD were 0.73 and 1.01%, respectively. The selectivity of the proposed method for protein was much higher than those of dye-binding and Lowry assays: Most common interferents to other protein assays such as tris, ethanolamine, deoxycholate, CsCl, citrate, and triton X-100 were tolerated at 100-fold concentrations in the analysis of 10mgl(-1) BSA, while the tolerance limits for other interferents, e.g., (NH(4))(2)SO(4) and acetylsalicylic acid (50-fold), SDS (25-fold), and glycerol (20-fold) were at acceptable levels. The redox reaction of Cu(II)-Nc as an outer-sphere electron transfer agent with the peptide bond and with four amino acid residues (cystine, cysteine, tryptophan, and tyrosine) was kinetically more favourable than that of Cu(II) alone in the biuret assay. Since the reduction product of Cu(II) with protein, i.e., Cu(I), was coordinatively saturated with Nc in the stable Cu(Nc)(2)(+) chelate, re-oxidation of the formed Cu(I) with Fenton-like reactions was not possible, thereby preventing a loss of chromophore. After conventional protein extraction, precipitation, and redissolution procedures, the protein contents of the minced meat

  7. Determination of nickel(II) at nanomolar levels using iodide-responsive gold-copper nanoparticles as colorimetric probes.

    Science.gov (United States)

    Xi, Hongyan; Liu, Qingyun; Chen, Zhengbo

    2018-01-08

    The authors present a colorimetric method for the quantification of Ni(II) at nanomolar levels. It is based on the use of iodide-responsive copper-gold nanoparticles (Cu-Au NPs) combined with the Ni(II)-catalyzed glutathione (GSH)-oxygen reaction system. In the presence of Ni(II), the catalytic reaction between GSH and oxygen is can triggered. This leads to the formation of GSSG which is bulky and hinders the access of iodide to the surface of the Cu-Au NPs. Concomitantly, the color of the solution containing the Cu-Au NPs changes from gray to red. Based on these findings, a method was developed for the quantitation of Ni(II) that has a detection limit as low as 0.54 nM. This is 1-3 orders of magnitude lower than that of previously reported optical methods. The assay has excellent selectivity for Ni(II), is rapid, cost-effective, portable, and allows for bare eye observation. Conceivably the method is suitable for field detection of Ni(II) in biological, food, and environmental samples. Graphical Abstract A sensitive colorimetric strategy for Ni(II) through the combination of iodide-responsive Cu-Au NPs with Ni(II)-catalyzed the oxidation of GSH by oxygen was presented.

  8. A curcumin-based TPA four-branched copper(II) complex probe for in vivo early tumor detection

    Energy Technology Data Exchange (ETDEWEB)

    Pi, Zongxin [Department of Chemical and Chemical Engineering, Hefei Normal University, Hefei 230001 (China); Wang, Jiafeng; Jiang, Bo [Department of Pharmacy, Anhui University of Chinese Medicine, Hefei 230038 (China); Cheng, Gang [Department of Chemical and Chemical Engineering, Hefei Normal University, Hefei 230001 (China); Zhou, Shuangsheng, E-mail: zshuangsheng@126.com [Department of Pharmacy, Anhui University of Chinese Medicine, Hefei 230038 (China); Center of Modern Experimental Technology, Anhui University, Hefei 230038 (China)

    2015-01-01

    A multibranched Cu(II) complex CuL{sub 2} curcumin-based was synthesized and characterized by single-crystal X-ray diffraction analysis. The photophysical properties of the complex have been investigated both experimentally and theoretically. The results show that the target complex exhibits higher quantum yield and larger two-photon absorption (TPA) cross-section in the near infrared (NIR) region compared with its free ligand. The cell imaging studies in vitro and in vivo reveal that the complex shows good photostability and excellent tumor targeting capability to tested cancerous cells, which can be potentially used for early tumor detection. - Graphical abstract: A multibranched Cu(II) complex was prepared from curcumin. The photophysical properties of the obtained complex have been investigated. The results exhibit that the complex has high capability to test cancerous cells and can distinguish between the cancerous and noncancerous cells, which should be potentially used for early tumor detection. - Highlights: • A novel multi-branched copper complex was synthesized. • The obtained compounds exhibited obvious TPA in high polar solvents. • The complex is a low toxicity at low-micromolar concentrations. • The complex exhibits larger TPA cross-section and brighter TPF imaging. • The complex has excellent targeting capability to tested cancerous cells.

  9. Extracellular proteins of Trametes hirsuta st. 072 induced by copper ions and a lignocellulose substrate.

    Science.gov (United States)

    Vasina, Daria V; Pavlov, Andrey R; Koroleva, Olga V

    2016-06-13

    Fungi are organisms with the highest natural capacity to degrade lignocellulose substrates, which is enabled by complex systems of extracellular enzymes, whose expression and secretion depend on the characteristics of substrates and the environment. This study reports a secretome analysis for white-rot basidiomycete Trametes hirsuta cultivated on a synthetic media and a lignocellulose substrate. We demonstrate that T. hirsuta st. 072 produces multiple extracellular ligninolytic, cellulolytic, hemicellulolytic, peroxide generating, and proteolytic enzymes, as well as cerato-platanins. In contrast to other white rot species described earlier, which mostly secreted glucanases and mannosidases in response to the presence of the lignocellulose substrate, T. hirsuta expressed a spectrum of extracellular cellulolytic enzymes containing predominantly cellobiases and xylanases. As proteomic analysis could not detect lignin peroxidase (LiP) among the secreted lignin degrading enzymes, we attributed the observed extracellular LiP - like activity to the expressed versatile peroxidase (VP). An accessory enzyme, glyoxal oxidase, was found among the proteins secreted in the media during submerged cultivation of T. hirsuta both in the presence and in the absence of copper. However, aryl-alcohol oxidase (AAO) was not identified, despite the presence of AAO enzymatic activity secreted by the fungus. The spectra of the expressed enzymes dramatically changed depending on the growth conditions. Transfer from submerged cultivation to surface cultivation with the lignocellulose substrate switched off expression of exo-β-1,3-glucanase and α-amylase and turned on secretion of endo-β-1,3-glucanase and a range of glycosidases. In addition, an aspartic peptidase started being expressed instead of family S53 protease. For the first time, we report production of cerato-platanin proteins by Trametes species. The secretion of cerato-platanins was observed only in response to contact with

  10. Mitochondrial DNA repair and replication proteins revealed by targeted chemical probes.

    Science.gov (United States)

    Wisnovsky, Simon; Jean, Sae Rin; Kelley, Shana O

    2016-07-01

    Efficient and accurate replication and repair of mitochondrial DNA is essential for cellular viability, yet only a minimal complement of mitochondrial proteins with relevant activities have been identified. Here, we describe an approach to screen for new pathways involved in the maintenance of mitochondrial DNA (mtDNA) that leverages the activities of DNA-damaging probes exhibiting specific subcellular localization. By conducting a siRNA screen of known nuclear DNA maintenance factors, and monitoring synergistic effects of gene depletion on the activity of mitochondria-specific DNA-damaging agents, we identify a series of proteins not previously recognized to act within mitochondria. These include proteins that function in pathways of oxidative DNA damage repair and dsDNA break repair, along with a novel mitochondrial DNA polymerase, POLθ, that facilitates efficient DNA replication in an environment prone to oxidative stress. POLθ expression levels affect the mutational rate of mitochondrial DNA, but this protein also appears critical for efficient mtDNA replication.

  11. NanoProbeArrays for the analysis of ultra-low-volume protein samples using piezoelectric liquid dispensing technology.

    Science.gov (United States)

    Nagaraj, Vinay Janthakahalli; Eaton, Seron; Wiktor, Peter

    2011-04-01

    Antibody microarrays are gaining popularity as a high-throughput technology to investigate the proteome. However, protein extracts from most body fluid or biopsy samples are available in very small volumes and are often unsuitable for large-scale antibody microarray studies. To demonstrate the potential for protein analysis with as little as a few nanoliters of sample, we have developed a new technology called NanoProbeArrays based on piezoelectric liquid dispensing for non-contact printing and probing of antibody arrays. Instead of flooding the protein sample on the antibody microarray surface, as in conventional microarray screening, a piezoelectric inkjet printer is used to dispense nanoliters of fluorescently labeled proteins over the antibody spots on the array. The ability of NanoProbeArrays to precisely identify and reliably distinguish between test proteins from different sources, without any loss of sensitivity and specificity as compared with conventional antibody microarrays, is illustrated here. The utility of NanoProbeArrays for biomarker identification in a complex biological sample was tested by detecting the cytokine interleukin-4 in serum. The significant reduction in volume of sample during NanoProbeArray analysis, as compared with conventional antibody microarrays, offers new opportunities for basic and applied proteomic research. Copyright © 2011 Society for Laboratory Automation and Screening. Published by Elsevier Inc. All rights reserved.

  12. EFFECTS OF HIGHER LEVELS OF CHROMIUM AND COPPER ON SOME HAEMATOLOGICAL PARAMETERS AND SERUM PROTEINS IN BROILERS

    Directory of Open Access Journals (Sweden)

    M. Tariq Javed, F, Ahmad. N, Z, Rafique1 and M, Bashir

    2003-01-01

    Full Text Available Effects of higher levels of chromium alone and in combination with copper were investigated in broiler chicks divided into seven equal groups viz. A, B, C, D, E, F and G. Group G served as control receiving no treatment. Groups A, B and F received chromium chloride at the rate of 2 g/kg and nicotinic acid 150 mg/kg feed while C, D and F received chromium chloride 8 g/kg and nicotinic acid 150mg/kg. Broilers of groups A and C received copper sulfate at the rate of 200 mg/kg while groups Band D 400 mg/kg feed. Haematological parameters studied revealed non-significant difference between treatment groups and control in haemoglobin concentration and total erythrocyte counts. However, only at 4th week, lower PCV was observed in birds fed higher levels of chromium chloride alone. Increase in TLC was observed in birds fed low chromium alone or' with low levels of copper. Results of serum proteins including total protein, albumin and globulin during first three weeks showed significantly or relatively lower values in treatment groups than control. Serum globulins generally revealed non-significant difference between treatment groups and control.

  13. Bodipy-FL-Verapamil: A Fluorescent Probe for the Study of Multidrug Resistance Proteins

    Directory of Open Access Journals (Sweden)

    Anna Rosati

    2004-01-01

    Full Text Available Most of the substances used as fluorescent probes to study drug transport and the effect of efflux blockers in multidrug resistant cells have many drawbacks, such as toxicity, unspecific background, accumulation in mitochondria. New fluorescent compounds, among which Bodipy‐FL‐verapamil (BV, have been therefore proposed as more useful tools. The uptake of BV has been evaluated by cytofluorimetry and fluorescence microscopy using cell lines that overexpress P‐glycoprotein (P388/ADR and LLC‐PK1/ADR or MRP (multidrug resistance‐related protein (PANC‐1 and clinical specimens from patients. The effect of specific inhibitors for P‐glycoprotein (verapamil and vinblastine or MRP (MK571 and probenecid has been also studied. BV intracellular concentrations were significantly lower in the two P‐glycoprotein overexpressing cell lines in comparison with the parental lines. In addition, verapamil and vinblastine increased the intracellular concentrations of the dye; MK571 and probenecid, two MRP inhibitors, increased BV levels in PANC‐1 cells, that express this protein. These findings were confirmed in clinical specimens from patients. Fluorescence microscopy revealed a faint fluorescence emission in P‐glycoprotein or MRP expressing cell lines; however, treatment with specific inhibitors significantly increased the fluorescence. BV is a useful tool for studying multidrug resistance proteins with different techniques such as cytofluorimetry and fluorescence microscopy, but does not discriminate between P‐glycoprotein and MRP. In comparison with other classic fluorescent probes, the assay with this dye is extremely rapid, simple, not toxic for cells, devoid of fluorescent background, and can be useful in the clinical settings.

  14. Limitations on the use of scanning probe microscopy for the measurement of field emission from copper surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Mizuno, Y.

    2004-02-25

    High electric-field breakdown in cm-wave x-band radio frequency (rf) accelerating structures is an important phenomenon limiting attainable accelerating gradients. Linear collider development requires an accelerating gradient of, at least, 70MeV/m (150MV/m surface electric field). The observed breakdown sequence usually consists of field emission (FE) from an electrically-conducting surface feature which heats the point of emission, thereby releasing gas from the surface and nearby bulk. The ionized gas makes a plasma that regeneratively heats the metal and releases more gas and electrons (via secondary emission) until a flashover occurs. The FE, however, occurs at a much lower surface electric field in experiments than predicted by theory. Experimental measurements of FE from macroscopic surface areas require the use of a geometrical field-enhancement factor, {beta}, to fit best the data to a Fowler-Nordheim (F-N) emission model with reasonable physical parameters. A newer, microscopic interpretation of the results proposes the existence of quasi-filamentary electrically-conducting channels between the metal bulk and the surface. These channels connect to emitting features near or on the surface, acting as microscopically field-enhanced electron emission sources. Analysis of emitter behavior suggesting that the macroscopic emission should primarily be due to classical geometrical-enhancement is, at least in some cases, due to the presence of these conducting nano-structures in the surface region. We describe an accelerator materials-related study of high electric field breakdown from polished copper (and other) surfaces, in dry-nitrogen gas ambient, using an atomic force microscope (AFM) modified to make F-N FE measurements. In early periods of this work, the results showed that the instrument might be capable of making F-N measurements on small surface areas of mechanically-polished and natively-oxidized high-quality copper surfaces to the GV/m level. The

  15. The interaction of taurine-salicylaldehyde Schiff base copper(II) complex with DNA and the determination of DNA using the complex as a fluorescence probe

    Science.gov (United States)

    Zhang, Xiaoyan; Wang, Yong; Zhang, Qianru; Yang, Zhousheng

    2010-09-01

    The interaction of taurine-salicylaldehyde Schiff base copper(II) (Cu(TSSB) 22+) complex with DNA was explored by using UV-vis, fluorescence spectrophotometry, and voltammetry. In pH 7.4 Tris-HCl buffer solution, the binding constant of the Cu(TSSB) 22+ complex interaction with DNA was 3.49 × 10 4 L mol -1. Moreover, due to the fluorescence enhancing of Cu(TSSB) 22+ complex in the presence of DNA, a method for determination of DNA with Cu(TSSB) 22+ complex as a fluorescence probe was developed. The fluorescence spectra indicated that the maximum excitation and emission wavelength were 389 nm and 512 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range of 0.03-9.03 μg mL -1 for calf thymus DNA (CT-DNA), 0.10-36 μg mL -1 for yeast DNA and 0.01-10.01 μg mL -1 for salmon DNA (SM-DNA), respectively. The corresponding detection limits are 7 ng mL -1 for CT-DNA, 3 ng mL -1 for yeast DNA and 3 ng mL -1 for SM-DNA. Using this method, DNA in synthetic samples was determined with satisfactory results.

  16. The interaction of taurine-salicylaldehyde Schiff base copper(II) complex with DNA and the determination of DNA using the complex as a fluorescence probe.

    Science.gov (United States)

    Zhang, Xiaoyan; Wang, Yong; Zhang, Qianru; Yang, Zhousheng

    2010-09-15

    The interaction of taurine-salicylaldehyde Schiff base copper(II) (Cu(TSSB)2(2+)) complex with DNA was explored by using UV-vis, fluorescence spectrophotometry, and voltammetry. In pH 7.4 Tris-HCl buffer solution, the binding constant of the Cu(TSSB)2(2+) complex interaction with DNA was 3.49 x 10(4) L mol(-1). Moreover, due to the fluorescence enhancing of Cu(TSSB)2(2+) complex in the presence of DNA, a method for determination of DNA with Cu(TSSB)2(2+) complex as a fluorescence probe was developed. The fluorescence spectra indicated that the maximum excitation and emission wavelength were 389 nm and 512 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range of 0.03-9.03 microg mL(-1) for calf thymus DNA (CT-DNA), 0.10-36 microg mL(-1) for yeast DNA and 0.01-10.01 microg mL(-1) for salmon DNA (SM-DNA), respectively. The corresponding detection limits are 7 ng mL(-1) for CT-DNA, 3 ng mL(-1) for yeast DNA and 3 ng mL(-1) for SM-DNA. Using this method, DNA in synthetic samples was determined with satisfactory results.

  17. Interactions between protein coated particles and polymer surfaces studied with the rotating particles probe.

    Science.gov (United States)

    Kemper, M; Spridon, D; van IJzendoorn, L J; Prins, M W J

    2012-05-29

    Nonspecific interactions between proteins and polymer surfaces have to be minimized in order to control the performance of biosensors based on immunoassays with particle labels. In this paper we investigate these nonspecific interactions by analyzing the response of protein coated magnetic particles to a rotating magnetic field while the particles are in nanometer vicinity to a polymer surface. We use the fraction of nonrotating (bound) particles as a probe for the interaction between the particles and the surface. As a model system, we study the interaction of myoglobin coated particles with oxidized polystyrene surfaces. We measure the interaction as a function of the ionic strength of the solution, varying the oxidation time of the polystyrene and the pH of the solution. To describe the data we propose a model in which particles bind to the polymer by crossing an energy barrier. The height of this barrier depends on the ionic strength of the solution and two interaction parameters. The fraction of nonrotating particles as a function of ionic strength shows a characteristic shape that can be explained with a normal distribution of energy barrier heights. This method to determine interaction parameters paves the way for further studies to quantify the roles of protein coated particles and polymers in their mutual nonspecific interactions in different matrixes.

  18. Visual detection of copper(II) ions in blood samples by controlling the leaching of protein-capped gold nanoparticles.

    Science.gov (United States)

    Lee, Yen-Fei; Deng, Ting-Wei; Chiu, Wei-Jane; Wei, Tsao-Yen; Roy, Prathik; Huang, Chih-Ching

    2012-04-21

    We have developed a simple, low-cost, paper-based probe for the selective colorimetric detection of copper ions (Cu(2+)) in aqueous solutions. The bovine serum albumin (BSA)-modified 13.3-nm Au nanoparticle (BSA-Au NP) probe was designed to detect Cu(2+) ions using lead ions (Pb(2+)) and 2-mercaptoethanol (2-ME) as leaching agents in a glycine-NaOH (pH 12.0) solution. In addition, a nitrocellulose membrane (NCM) was used to trap the BSA-Au NPs, leading to the preparation of a nanocomposite film consisting of a BSA-Au NP-decorated membrane (BSA-Au NPs/NCM). The BSA-Au NPs probe operates on the principle that Cu deposition on the surface of the BSA-Au NPs inhibits their leaching ability, which is accelerated by Pb(2+) ions in the presence of 2-ME. Under optimal solution conditions (5 mM glycine-NaOH (pH 12.0), Pb(2+) (50 μM), and 2-ME (1.0 M)), the Pb(2+)/2-ME-BSA-Au NPs/NCM enabled the detection of Cu(2+) at nanomolar concentrations in aqueous solutions by the naked eye with high selectivity (at least 100-fold over other metal ions). In addition, this cost-effective probe allowed for the rapid and simple determination of Cu(2+) ions in not only natural water samples but also in a complex biological sample (in this case, blood sample).

  19. Posttranslational regulation of copper transporters

    NARCIS (Netherlands)

    van den Berghe, P.V.E.

    2009-01-01

    The transition metal copper is an essential cofactor for many redox-active enzymes, but excessive copper can generate toxic reactive oxygen species. Copper homeostasis is maintained by highly conserved proteins, to balance copper uptake, distribution and export on the systemic and cellular level.

  20. [Sensitive method for continuous detection of peptides and proteins using the biuret reaction and a copper-Sephadex reactor (author's transl)].

    Science.gov (United States)

    Later, R; Quincy, C

    1979-06-01

    We describe a detection method relying both on the copper displacement from a Sephadex gel by peptides and proteins, and on the subsequent colorimetric determination of the complexed copper. The system described is fully automated and it permits a continuous analysis of column effluents. The choice of cuprizone as a detecting reagent for copper, enables one to bring the detection limit down to 200 ng for albumin and 60 ng for alanylglycylglycin. The specificity of the method is the same as the biuret reaction. Some examples of the possible applications are given.

  1. Quantitative Characterization of Bivalent Probes for a Dual Bromodomain Protein, Transcription Initiation Factor TFIID Subunit 1.

    Science.gov (United States)

    Suh, Junghyun L; Watts, Brian; Stuckey, Jacob I; Norris-Drouin, Jacqueline L; Cholensky, Stephanie H; Dickson, Bradley M; An, Yi; Mathea, Sebastian; Salah, Eidarus; Knapp, Stefan; Khan, Abid; Adams, Alexander T; Strahl, Brian D; Sagum, Cari A; Bedford, Mark T; James, Lindsey I; Kireev, Dmitri B; Frye, Stephen V

    2018-03-28

    Multivalent binding is an efficient means to enhance the affinity and specificity of chemical probes targeting multidomain proteins in order to study their function and role in disease. While the theory of multivalent binding is straightforward, physical and structural characterization of bivalent binding encounters multiple technical difficulties. We present a case study where a combination of experimental techniques and computational simulations was used to comprehensively characterize the binding and structure-affinity relationships for a series of Bromosporine-based bivalent bromodomain ligands with a bivalent protein, Transcription Initiation Factor TFIID subunit 1 (TAF1). Experimental techniques-Isothermal Titration Calorimetry, X-ray Crystallography, Circular Dichroism, Size Exclusion Chromatography-Multi-Angle Light Scattering, and Surface Plasmon Resonance-were used to determine structures, binding affinities, and kinetics of monovalent ligands and bivalent ligands with varying linker lengths. The experimental data for monomeric ligands were fed into explicit computational simulations, in which both ligand and protein species were present in a broad range of concentrations, and in up to a 100 s time regime, to match experimental conditions. These simulations provided accurate estimates for apparent affinities (in good agreement with experimental data), individual dissociation microconstants and other microscopic details for each type of protein-ligand complex. We conclude that the expected efficiency of bivalent ligands in a cellular context is difficult to estimate by a single technique in vitro, due to higher order associations favored at the concentrations used, and other complicating processes. Rather, a combination of structural, biophysical, and computational approaches should be utilized to estimate and characterize multivalent interactions.

  2. Environment of copper in Pseudomonas aeruginosa azurin probed by binding of exogenous ligands to Met121X (X = Gly, Ala, Val, Leu, or Asp) mutants.

    Science.gov (United States)

    Bonander, N; Karlsson, B G; Vänngård, T

    1996-02-20

    The binding of small exogenous ligands to mutants of the blue copper protein azurin from Pseudomonas aeruginosa, altered in the axial position, Met121X (X = Gly, Ala, Val, Leu, or Asp), has been studied with optical and electron paramagnetic resonance (EPR) spectroscopy. The results show that small molecules can enter the pocket left by the side chain of Met121. For azide, the dissociation constants are Leu > Val > Ala, reflecting the increasing space available. The Gly and Asp mutants bind azide less strongly than the Ala mutant, due to competition with water (Gly) and the polar side chain (Asp). Similar trends are found for thiocyanate. Cyanide binds equally well to the Ala and Val mutants. A number of other small potential ligands were tried. Alcohols do not affect room-temperature optical spectra, but at low temperatures, the EPR spectrum is stellacyanin-like, indicative of a weak axial interaction. Ligands binding with a carboxyl group or nitrogen (e.g. acetate or azide) convert the metal center to a form intermediate between regular types 1 and 2, presumably by pulling the copper ion out of the trigonal plane formed by Cys(S) and two His(N). Cyanide interacts strongly as shown by the hyperfine coupling to the 13C nucleus. With increasing strength of the axial interaction, the two major bands in the visible region (600 and 400-500 nm) shift in parallel to higher energy, and at the same time, the strength of the latter transition increases at the expense of the former. This demonstrates that these transitions have a common origin, namely S-to-Cu charge transfer transition.

  3. Ordered Assembly and Controlled Electron Transfer of the Blue Copper Protein Azurin at Gold (111) Single-Crystal Substrates

    DEFF Research Database (Denmark)

    Chi, Qijin; Zhang, Jingdong; Andersen, Jens Enevold Thaulov

    2001-01-01

    or monolayers. In this orientation mode azurin molecules on Au(111) are oriented with the redox center (copper atom) facing the electrode surface. This is opposite to the orientation of azurin on bare gold which is via a surface disulfide group such as recently reported. Scanning tunneling microscopy (STM......) with molecular resolution reveals that both well-ordered alkanethiol and protein adlayers are present. Adsorbed azurin molecules exhibit high stability and retain electron transfer (ET) function. Long-range interfacial ET between azurin and Au(111) across variable-length alkanethiol bridges was systematically...

  4. Evaluating Protein Structure and Dynamics Using Co-Solvents, Photochemical Triggers, and Site-Specific Spectroscopic Probes

    Science.gov (United States)

    Abaskharon, Rachel M.

    As ubiquitous and diverse biopolymers, proteins are dynamic molecules that are constantly engaging in inter- and intramolecular interactions responsible for their structure, fold, and function. Because of this, gaining a comprehensive understanding of the factors that control protein conformation and dynamics remains elusive as current experimental techniques often lack the ability to initiate and probe a specific interaction or conformational transition. For this reason, this thesis aims to develop methods to control and monitor protein conformations, conformational transitions, and dynamics in a site-specific manner, as well as to understand how specific and non-specific interactions affect the protein folding energy landscape. First, by using the co-solvent, trifluoroethanol (TFE), we show that the rate at which a peptide folds can be greatly impacted and thus controlled by the excluded volume effect. Secondly, we demonstrate the utility of several light-responsive molecules and reactions as methods to manipulate and investigate protein-folding processes. Using an azobenzene linker as a photo-initiator, we are able to increase the folding rate of a protein system by an order of magnitude by channeling a sub-population through a parallel, faster folding pathway. Additionally, we utilize a tryptophan-mediated electron transfer process to a nearby disulfide bond to strategically unfold a protein molecule with ultraviolet light. We also demonstrate the potential of two ruthenium polypyridyl complexes as ultrafast phototriggers of protein reactions. Finally, we develop several site-specific spectroscopic probes of protein structure and environment. Specifically, we demonstrate that a 13C-labeled aspartic acid residue constitutes a useful site-specific infrared probe for investigating salt-bridges and hydration dynamics of proteins, particularly in proteins containing several acidic amino acids. We also show that a proline-derivative, 4-oxoproline, possesses novel

  5. One gene, two proteins: coordinated production of a copper chaperone by differential transcript formation and translational frameshifting in Escherichia coli.

    Science.gov (United States)

    Drees, Steffen L; Klinkert, Birgit; Helling, Stefan; Beyer, Dominik F; Marcus, Katrin; Narberhaus, Franz; Lübben, Mathias

    2017-11-01

    Programmed ribosomal frameshifting (PRF) is a translational anomaly causing the ribosome to shift into an alternative reading frame. PRFs are common in viral genomes, using a single nucleotide sequence to code for two proteins in overlapping frames. In bacteria and eukaryota, PRFs are less frequent. We report on a PRF in the copper detoxification system of Escherichia coli where a metallochaperone is generated out of the first 69 amino acids and a C-terminal out-of-frame glycine of the gene copA. copA besides codes for the P 1B -ATPase CopA, a membrane-integral protein and principal interaction target of the chaperone. To enhance the production of the frameshift-generated cytosolic copper binding protein a truncated transcript is produced from the monocistronic copA gene. This shorter transcript is essential for producing sufficient amounts of the chaperone to support the membrane pump. The findings close the gap in our understanding of the molecular physiology of cytoplasmic copper transport in E. coli, revealing that a chaperone-like entity is required for full functionality of the P 1B -ATPase copper pump. We, moreover, demonstrate that the primary transcriptional response to copper results in formation of the small transcript and concurrently, the metallochaperone plays a key role in resistance against copper shock. © 2017 John Wiley & Sons Ltd.

  6. Direct protein quantification in complex sample solutions by surface-engineered nanorod probes

    KAUST Repository

    Schrittwieser, Stefan

    2017-06-30

    Detecting biomarkers from complex sample solutions is the key objective of molecular diagnostics. Being able to do so in a simple approach that does not require laborious sample preparation, sophisticated equipment and trained staff is vital for point-of-care applications. Here, we report on the specific detection of the breast cancer biomarker sHER2 directly from serum and saliva samples by a nanorod-based homogeneous biosensing approach, which is easy to operate as it only requires mixing of the samples with the nanorod probes. By careful nanorod surface engineering and homogeneous assay design, we demonstrate that the formation of a protein corona around the nanoparticles does not limit the applicability of our detection method, but on the contrary enables us to conduct in-situ reference measurements, thus further strengthening the point-of-care applicability of our method. Making use of sandwich assays on top of the nanorods, we obtain a limit of detection of 110 pM and 470 pM in 10-fold diluted spiked saliva and serum samples, respectively. In conclusion, our results open up numerous applications in direct protein biomarker quantification, specifically in point-of-care settings where resources are limited and ease-of-use is of essence.

  7. Tracking G-protein-coupled receptor activation using genetically encoded infrared probes.

    Science.gov (United States)

    Ye, Shixin; Zaitseva, Ekaterina; Caltabiano, Gianluigi; Schertler, Gebhard F X; Sakmar, Thomas P; Deupi, Xavier; Vogel, Reiner

    2010-04-29

    Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.

  8. Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Chalmers, Michael J; Busby, Scott A; Pascal, Bruce D; He, Yuanjun; Hendrickson, Christopher L; Marshall, Alan G; Griffin, Patrick R

    2006-02-15

    Amide hydrogen/deuterium exchange is a powerful biophysical technique for probing changes in protein dynamics induced by ligand interaction. The inherent low throughput of the technology has limited its impact on drug screening and lead optimization. Automation increases the throughput of H/D exchange to make it compatible with drug discovery efforts. Here we describe the first fully automated H/D exchange system that provides highly reproducible H/D exchange kinetics from 130 ms to 24 h. Throughput is maximized by parallel sample processing, and the system can run H/D exchange assays in triplicate without user intervention. We demonstrate the utility of this system to differentiate structural perturbations in the ligand-binding domain (LBD) of the nuclear receptor PPARgamma induced upon binding a full agonist and a partial agonist. PPARgamma is the target of glitazones, drugs used for treatment of insulin resistance associated with type II diabetes. Recently it has been shown that partial agonists of PPARgamma have insulin sensitization properties while lacking several adverse effects associated with full agonist drugs. To further examine the mechanism of partial agonist activation of PPARgamma, we extended our studies to the analysis of ligand interactions with the heterodimeric complex of PPARgamma/RXRalpha LBDs. To facilitate analysis of H/D exchange of large protein complexes, we performed the experiment with a 14.5-T Fourier transform ion cyclotron resonance mass spectrometer capable of measuring mass with accuracy in the ppb range.

  9. Probing Adsorption / Desorption Processes at the Liquid / Solid Interface: Thiols and Proteins

    Science.gov (United States)

    Campbell, Charles; Jung, Linda S.; Shumaker-Parry, Jennifer; Nelsen, K. E.; Stayton, P. S.; Gelb, M. H.; Aebersold, R.

    2001-03-01

    surface template that can be used to probe the kinetics and equilibrium binding constants for: (1) peripheral membrane proteins binding to vesicle walls, and (2) the binding of DNA-binding proteins to select oligonucleotide sequences. By detecting the reflected light at a high-contrast angle with spatial resolution using a simple CCD camera, these SPR measurements can be performed quite simply in a microscopy mode to probe the distribution of species across the surface.

  10. Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein.

    Science.gov (United States)

    Balogh, Ria K; Gyurcsik, Béla; Hunyadi-Gulyás, Éva; Christensen, Hans E M; Jancsó, Attila

    2016-07-01

    Metal ion regulation is essential for living organisms. In prokaryotes metal ion dependent transcriptional factors, the so-called metalloregulatory proteins play a fundamental role in controlling the concentration of metal ions. These proteins recognize metal ions with an outstanding selectivity. A detailed understanding of their function may be exploited in potential health, environmental and analytical applications. Members of the MerR protein family sense a broad range of mostly late transition and heavy metal ions through their cysteine thiolates. The air sensitivity of latter groups makes the expression and purification of such proteins challenging. Here we describe a method for the purification of the copper-regulatory CueR protein under optimized conditions. In order to avoid protein precipitation and/or eventual aggregation and to get rid of the co-purifying Escherichia coli elongation factor, our procedure consisted of four steps supplemented by DNA digestion. Subsequent anion exchange on Sepharose FF Q 16/10, affinity chromatography on Heparin FF 16/10, second anion exchange on Source 30 Q 16/13 and gel filtration on Superdex 75 26/60 resulted in large amounts of pure CueR protein without any affinity tag. Structure and functionality tests performed with mass spectrometry, circular dichroism spectroscopy and electrophoretic gel mobility shift assays approved the success of the purification procedure. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Short-Term Protein Stable Isotope Probing of Microbial Communities to Associate Functions with Taxa (Invited)

    Science.gov (United States)

    Lipton, M. S.; Slysz, G. W.; Steinke, L. A.; Ward, D. M.; Klatt, C. G.; Clauss, T. R.; Purvine, S. O.; Anderson, G. A.; Payne, S. H.; Bryant, D. A.

    2013-12-01

    Determining which taxa in a community perform which functions is essential for understanding metabolite fluxes and metabolic interactions among community members. Specific taxa will alter their metabolism in order to acclimate to changing environmental factors such as light through the diel cycle, changing temperature and other factors. Monitoring which proteins are being expressed, and the quantitative protein expression patterns in the individual taxa as a response to external stimuli is key to understanding these mechanisms. Protein stable isotope probing (Pro-SIP) has strong potential for revealing key metabolizing taxa in complex microbial communities. In Pro-SIP studies, label incorporation is determined by the extent of the change in the isotopic profile of peptides when measured by mass spectrometry. While most Pro-SIP work to date has been performed under controlled laboratory conditions to allow extensive isotope labeling of the target organism(s), these techniques have not been applied to short term in situ studies due to the small degree of partial labeling of the proteins. We have applied Pro-SIP to study the assimilation of a labeled substrate into proteins to determine which taxa are responsible for sequestration of dissolved inorganic carbon in microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park. This community is fueled by sunlight as it transitions from dark to light; the aim was to understand the light-dependent pathway of inorganic carbon incorporation into different taxa during the early morning hours when the mat was in low light and anoxic. Each mat sample was incubated with 13C-bicarbonate for 3 h. Substrate assimilation was determined through standard proteomic techniques along with the use of SIPPER, a collection of algorithms that sensitively measure small changes in peptide isotopic patterns, allowing the determination of which taxa assimilated the substrate during this period. For the

  12. Probing the role of backbone hydrogen bonds in protein-peptide interactions by amide-to-ester mutations

    DEFF Research Database (Denmark)

    Eildal, Jonas N N; Hultqvist, Greta; Balle, Thomas

    2013-01-01

    of the protein ligand, the role of the backbone hydrogen bonds in the binding reaction is not known. Using amide-to-ester substitutions to perturb the backbone hydrogen-bonding pattern, we have systematically probed putative backbone hydrogen bonds between four different PDZ domains and peptides corresponding...

  13. A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags.

    Science.gov (United States)

    Söveges, B; Imre, T; Szende, T; Póti, Á L; Cserép, G B; Hegedűs, T; Kele, P; Németh, K

    2016-07-07

    Fluorescent tagging of proteins via accessible cysteine residues is of paramount importance. In this study, model proteins of interest (mitogen-activated protein kinases) were labeled successfully in native state on their free thiols by direct fluorescence derivatization, or in a sequential manner where conjugation of the site specific linker and the fluorophore is carried out in two steps. To this end we designed and prepared two novel chemical reporters carrying vinyl sulfone as Cys targeting function and cyclooctyne motifs, suitable for subsequent conjugation with fluorogenic azides via copper free strain-promoted azide-alkyne click chemistry. Direct and sequential labeling reaction steps were analyzed by native PAGE, capillary zone electrophoresis and tandem mass spectrometry. The efficiency of tagging was correlated with solvent accessibility of the Cys residues. Our results indicated that conjugation of native proteins by vinyl sulfone linkers was fast and thiol-selective. Subsequent click reaction with fluorogenic dyes generates intensive fluorescence signals and fulfills all requirements of bioorthogonality.

  14. Determination of the electron self-exchange rates of blue copper proteins by super-WEFT NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Ma Lixin; Philipp, Else; Led, Jens J. [University of Copenhagen, H.C. Orsted Institute, Department of Chemistry (Denmark)

    2001-03-15

    An NMR approach for determining the electron self-exchange (ESE) rate constants in blue copper proteins is presented. The approach uses the paramagnetic relaxation enhancement of resonances in 1D {sup 1}H super-WEFT spectra of partly oxidized (paramagnetic) proteins. These spectra allow a more precise determination of the relevant paramagnetic linebroadenings than conventional 1D {sup 1}H spectra and, thus, permit a more detailed investigation of the applicability of the linebroadenings for determining the electron exchange rates. The approach was used to estimate the ESE rate constant of plastocyanin from Anabaena variabilis. It was found that, although the rate constant can be determined accurately from a series of resonances, precise but erroneous constants are obtained from the resonances of the copper-bound residues, unless a narrow splitting of these resonances caused by the presence of two conformations is taken into account. As demonstrated here, this complication can be overcome by a correct analysis of the paramagnetic broadening of the combined double signals. Because of the high resolution and specific sensitivity of the approach it should be generally applicable to estimate electron transfer rates, k, if the paramagnetic relaxation enhancement R{sub 2p} of the resonances can be determined, and the conditions k<<>k>>R{sub 2p} are fulfilled, {delta}{omega}{sub p} being the frequency separation between corresponding diamagnetic and paramagnetic sites.

  15. Copper resistance determinants in bacteria.

    Science.gov (United States)

    Brown, N L; Rouch, D A; Lee, B T

    1992-01-01

    Copper is an essential trace element that is utilized in a number of oxygenases and electron transport proteins, but it is also a highly toxic heavy metal, against which all organisms must protect themselves. Known bacterial determinants of copper resistance are plasmid-encoded. The mechanisms which confer resistance must be integrated with the normal metabolism of copper. Different bacteria have adopted diverse strategies for copper resistance, and this review outlines what is known about bacterial copper resistance mechanisms and their genetic regulation.

  16. Analysis of sequencing data for probing RNA secondary structures and protein-RNA binding in studying posttranscriptional regulations.

    Science.gov (United States)

    Hu, Xihao; Wu, Yang; Lu, Zhi John; Yip, Kevin Y

    2016-11-01

    High-throughput sequencing has been used to study posttranscriptional regulations, where the identification of protein-RNA binding is a major and fast-developing sub-area, which is in turn benefited by the sequencing methods for whole-transcriptome probing of RNA secondary structures. In the study of RNA secondary structures using high-throughput sequencing, bases are modified or cleaved according to their structural features, which alter the resulting composition of sequencing reads. In the study of protein-RNA binding, methods have been proposed to immuno-precipitate (IP) protein-bound RNA transcripts in vitro or in vivo By sequencing these transcripts, the protein-RNA interactions and the binding locations can be identified. For both types of data, read counts are affected by a combination of confounding factors, including expression levels of transcripts, sequence biases, mapping errors and the probing or IP efficiency of the experimental protocols. Careful processing of the sequencing data and proper extraction of important features are fundamentally important to a successful analysis. Here we review and compare different experimental methods for probing RNA secondary structures and binding sites of RNA-binding proteins (RBPs), and the computational methods proposed for analyzing the corresponding sequencing data. We suggest how these two types of data should be integrated to study the structural properties of RBP binding sites as a systematic way to better understand posttranscriptional regulations. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  17. Differential Mobility Spectrometry-Hydrogen Deuterium Exchange (DMS-HDX) as a Probe of Protein Conformation in Solution

    Science.gov (United States)

    Zhu, Shaolong; Campbell, J. Larry; Chernushevich, Igor; Le Blanc, J. C. Yves; Wilson, Derek J.

    2016-06-01

    Differential mobility spectrometry (DMS) is an ion mobility technique that has been adopted chiefly as a pre-filter for small- to medium-sized analytes (hydrogen deuterium exchange (DMS-HDX) to probe the gas-phase conformations generated from proteins that were initially folded, partially-folded, and unfolded in solution. Our findings indicate that proteins with distinct structural features in solution exhibit unique deuterium uptake profiles as function of their optimal transmission through the DMS. Ultimately we propose that DMS-HDX can, if properly implemented, provide rapid measurements of liquid-phase protein structural stability that could be of use in biopharmaceuticals development.

  18. Hydrogen bonding of sulfur ligands in blue copper and iron-sulfur proteins: detection by resonance raman spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Mino, Y.; Loehr, T.M.; Wada, K.; Matsubara, H.; Sanders-Loehr, J.

    1987-12-15

    The resonance Raman spectrum of the blue copper protein azurin from Alcaligenes denitrificans exhibits nine vibrational modes between 330 and 460 cm/sup -1/, seven of which shift 0.4-3.0 cm/sup -1/ to lower energy after incubation of the protein in D/sub 2/O. These deuterium-dependent shifts have been previously ascribed to exchangeable protons on imidazole ligands or to exchangeable protons on amide groups which are hydrogen bonded to the cysteine thiolate ligands (a feature common to all blue copper proteins of known structure). In order to distinguish between these two possibilities, a systematic investigation of Fe/sub 2/S/sub 2/(Cys)/sub 4/-containing proteins was undertaken. Extensive hydrogen bonding between sulfur ligands and the polypeptide backbone had been observed in the crystal structure of ferredoxin from Spirulina platensis. The resonance Raman spectrum of this protein is typical of a chloroplast-type ferredoxin and exhibits deuterium-dependent shifts of -0.3 to -0.5 cm/sup -1/ in the Fe-S modes at 283, 367, and 394 cm/sup -1/ and -0.6 to -0.8 cm/sup -1/ in the Fe-S modes at 328 and 341 cm/sup -1/. Considerably greater deuterium sensitivity is observed in the Raman spectra of spinach ferredoxin and bovine adrenodoxin, particularly for the symmetric stretching vibration of the Fe/sub 2/S/sub 2/ moiety at approx. 390 cm/sup -1/. This feature decreases of 9.8 and 1.1 cm/sup -1/, respectively, for the two oxidized proteins in D/sub 2/O and by 1.8 cm/sup -1/ for reduced adrenodoxin in D/sub 2/O. These results suggest that the bridging sulfido groups may be more extensively hydrogen bonded in spinach ferredoxin and adrenodoxin than in S. platensis ferredoxin, with a further increase in hydrogen-bond strength in the reduced form of adrenodoxin.

  19. Theoretical Simulations and Ultrafast Pump-probe Spectroscopy Experiments in Pigment-protein Photosynthetic Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Buck, D. R. [Iowa State Univ., Ames, IA (United States)

    2000-09-12

    Theoretical simulations and ultrafast pump-probe laser spectroscopy experiments were used to study photosynthetic pigment-protein complexes and antennae found in green sulfur bacteria such as Prosthecochloris aestuarii, Chloroflexus aurantiacus, and Chlorobium tepidum. The work focused on understanding structure-function relationships in energy transfer processes in these complexes through experiments and trying to model that data as we tested our theoretical assumptions with calculations. Theoretical exciton calculations on tubular pigment aggregates yield electronic absorption spectra that are superimpositions of linear J-aggregate spectra. The electronic spectroscopy of BChl c/d/e antennae in light harvesting chlorosomes from Chloroflexus aurantiacus differs considerably from J-aggregate spectra. Strong symmetry breaking is needed if we hope to simulate the absorption spectra of the BChl c antenna. The theory for simulating absorption difference spectra in strongly coupled photosynthetic antenna is described, first for a relatively simple heterodimer, then for the general N-pigment system. The theory is applied to the Fenna-Matthews-Olson (FMO) BChl a protein trimers from Prosthecochloris aestuarii and then compared with experimental low-temperature absorption difference spectra of FMO trimers from Chlorobium tepidum. Circular dichroism spectra of the FMO trimer are unusually sensitive to diagonal energy disorder. Substantial differences occur between CD spectra in exciton simulations performed with and without realistic inhomogeneous distribution functions for the input pigment diagonal energies. Anisotropic absorption difference spectroscopy measurements are less consistent with 21-pigment trimer simulations than 7-pigment monomer simulations which assume that the laser-prepared states are localized within a subunit of the trimer. Experimental anisotropies from real samples likely arise from statistical averaging over states with diagonal energies shifted by

  20. New insights into metal interactions with the prion protein: EXAFS analysis and structure calculations of copper binding to a single octarepeat from the prion protein.

    Science.gov (United States)

    McDonald, Alex; Pushie, M Jake; Millhauser, Glenn L; George, Graham N

    2013-11-07

    Copper coordination to the prion protein (PrP) has garnered considerable interest for almost 20 years, due in part to the possibility that this interaction may be part of the normal function of PrP. The most characterized form of copper binding to PrP has been Cu(2+) interaction with the conserved tandem repeats in the N-terminal domain of PrP, termed the octarepeats, with many studies focusing on single and multiple repeats of PHGGGWGQ. Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used in several previous instances to characterize the solution structure of Cu(2+) binding into the peptide backbone in the HGGG portion of the octarepeats. All previous EXAFS studies, however, have benefitted from crystallographic structure information for [Cu(II) (Ac-HGGGW-NH2)(-2H)] but have not conclusively demonstrated that the complex EXAFS spectrum represents the same coordination environment for Cu(2+) bound to the peptide backbone. Density functional structure calculations as well as full multiple scattering EXAFS curve fitting analysis are brought to bear on the predominant coordination mode for Cu(2+) with the Ac-PHGGGWGQ-NH2 peptide at physiological pH, under high Cu(2+) occupancy conditions. In addition to the structure calculations, which provide a thermodynamic link to structural information, methods are also presented for extensive deconvolution of the EXAFS spectrum. We demonstrate how the EXAFS data can be analyzed to extract the maximum structural information and arrive at a structural model that is significantly improved over previous EXAFS characterizations. The EXAFS spectrum for the chemically reduced form of copper binding to the Ac-PHGGGWGQ-NH2 peptide is presented, which is best modeled as a linear two-coordinate species with a single His imidazole ligand and a water molecule. The extent of in situ photoreduction of the copper center during standard data collection is also presented, and EXAFS curve fitting of the photoreduced species

  1. Cytochrome b561, copper, β-cleaved amyloid precursor protein and niemann-pick C1 protein are involved in ascorbate-induced release and membrane penetration of heparan sulfate from endosomal S-nitrosylated glypican-1.

    Science.gov (United States)

    Cheng, Fang; Fransson, Lars-Åke; Mani, Katrin

    2017-11-15

    Ascorbate-induced release of heparan sulfate from S-nitrosylated heparan sulfate proteoglycan glypican-1 takes place in endosomes. Heparan sulfate penetrates the membrane and is transported to the nucleus. This process is dependent on copper and on expression and processing of the amyloid precursor protein. It remains unclear how exogenously supplied ascorbate can generate HS-anMan in endosomes and how passage through the membrane is facilitated. Here we have examined wild-type, Alzheimer Tg2576 and amyloid precursor protein (-/-) mouse fibroblasts and human fetal and Niemann-Pick C1 fibroblasts by using deconvolution immunofluorescence microscopy, siRNA technology and [S 35 ]sulfate-labeling, vesicle isolation and gel chromatography. We found that ascorbate-induced release of heparan sulfate was dependent on expression of endosomal cytochrome b561. Formation and nuclear transport of heparan sulfate was suppressed by inhibition of β-processing of the amyloid precursor protein and formation was restored by copper (I) ions. Membrane penetration was not dependent on amyloid beta channel formation. Inhibition of endosomal exit resulted in accumulation of heparan sulfate in vesicles that exposed the C-terminal of the amyloid precursor protein externally. Endosome-to-nucleus transport was also dependent on expression of the Niemann-Pick C1 protein. We propose that ascorbate is taken up from the medium and is oxidized by cytochrome b561 which, in turn, reduces copper (II) to copper (I) present in the N-terminal, β-cleaved domain of the amyloid precursor protein. Re-oxidation of copper (I) is coupled to reductive, deaminative release of heparan sulfate from glypican-1. Passage through the membrane may be facilitated by the C-terminal, β-cleaved fragment of the amyloid precursor protein and the Niemann-Pick C1 protein. Copyright © 2017. Published by Elsevier Inc.

  2. Joint Toxicity of Arsenic, Copper and Glyphosate on Behavior, Reproduction and Heat Shock Protein Response in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Yunbiao; Ezemaduka, Anastasia N; Li, Zhuheng; Chen, Zhanyan; Song, Chuantao

    2017-04-01

    The soil nematode Caenorhabditis elegans was used in 24-h acute exposures to arsenic (As), copper (Cu) and glyphosate (GPS) and to mixtures of As/Cu and As/GPS to investigate the effects of mixture exposures in the worms. A synergistic type of interaction was observed for acute toxicity with the As/Cu and As/GPS mixtures. Sublethal 24-h exposures of 1/1000, 1/100 and 1/10 of the LC50 concentrations for As, Cu and GPS individually and for As/Cu and As/GPS mixtures were conducted to observe responses in locomotory behavior (head thrashing), reproduction, and heat shock protein expression. Head thrash frequency and reproduction exhibited concentration dependent decreases in both individual and combined exposures to the tested chemical stressors, and showed synergistic interactions even at micromolar concentrations. Furthermore, the HSP70 protein level was significantly increased following exposure to individual and combined chemical stressors in wild-type C. elegans. Our findings establish for the first time the effects of exposure to As/GPS and As/Cu mixtures in C. elegans.

  3. Simplified sample preparation method for protein identification by matrix-assisted laser desorption/ionization mass spectrometry: in-gel digestion on the probe surface

    DEFF Research Database (Denmark)

    Stensballe, A; Jensen, Ole Nørregaard

    2001-01-01

    /ionization-time of flight mass spectrometry (MALDI-TOF-MS) is used as the first protein screening method in many laboratories because of its inherent simplicity, mass accuracy, sensitivity and relatively high sample throughput. We present a simplified sample preparation method for MALDI-MS that enables in-gel digestion...... for protein identification similar to that obtained by the traditional protocols for in-gel digestion and MALDI peptide mass mapping of human proteins, i.e. approximately 60%. The overall performance of the novel on-probe digestion method is comparable with that of the standard in-gel sample preparation...... of protein samples directly on the MALDI-MS metal probe. Removal of detergent and reagents as well as protein reduction and S-alkylation were performed prior to cutting of protein samples from the polyacrylamide gel slab. The general utility of this approach was demonstrated by on-probe digestion and MALDI...

  4. Design and synthesis of new fluorescent probe for rapid and highly sensitive detection of proteins via electrophoretic gel stain.

    Science.gov (United States)

    Suzuki, Yoshio; Takagi, Nobuyuki; Chimuro, Tomoyuki; Shinohara, Atsushi; Sakaguchi, Nao; Hiratsuka, Atsunori; Yokoyama, Kenji

    2011-06-01

    A new fluorescent molecular probe, 2,2'-(1E,1'E)-2,2'-(4-(dicyanomethylene)-4H-pyrane-2,6-diyl)bis(ethene-2,1-diyl)bis(sodium benzenesulfonate) salt (1), possessing the cyanopyranyl moieties and two benzene sulfonic acid groups was designed and synthesized to detect proteins in solution and for high-throughput SDS-PAGE. Compound 1 exhibited no fluorescence in the absence of proteins; however, it exhibited strong fluorescence on the addition of bovine serum albumin as a result of intramolecular charge transfer. Compared with the conventional protocols for in-gel protein staining, such as SYPRO Ruby and silver staining, 1 achieves higher sensitivity, even though it offers a simplified, higher throughput protocol. In fact, the total time required for protein staining was 60-90 min under optimum conditions much shorter than that required by the less-sensitive silver staining or SYPRO Ruby staining protocols. Moreover, 1 was successfully applied to protein identification by mass spectrometry via in-gel tryptic digestion, Western blotting, and native PAGE together with protein staining by 1, which is a modified protocol of blue native PAGE (BN-PAGE). Thus, 1 may facilitate high-sensitivity protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Differential Mobility Spectrometry-Hydrogen Deuterium Exchange (DMS-HDX) as a Probe of Protein Conformation in Solution.

    Science.gov (United States)

    Zhu, Shaolong; Campbell, J Larry; Chernushevich, Igor; Le Blanc, J C Yves; Wilson, Derek J

    2016-06-01

    Differential mobility spectrometry (DMS) is an ion mobility technique that has been adopted chiefly as a pre-filter for small- to medium-sized analytes (DMS-field asymmetric waveform ion mobility spectroscopy (FAIMS)-the application of DMS to intact biomacromolecules remains largely unexplored. In this work, we employ DMS combined with gas-phase hydrogen deuterium exchange (DMS-HDX) to probe the gas-phase conformations generated from proteins that were initially folded, partially-folded, and unfolded in solution. Our findings indicate that proteins with distinct structural features in solution exhibit unique deuterium uptake profiles as function of their optimal transmission through the DMS. Ultimately we propose that DMS-HDX can, if properly implemented, provide rapid measurements of liquid-phase protein structural stability that could be of use in biopharmaceuticals development. Graphical Abstract ᅟ.

  6. Copper Complexes with Non-innocent Ligands: Probing Cu-II/catecholato-Cu-I/o-Semiquinonato Redox Isomer Equilibria with EPR Spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Kaim, W.; Wanner, M.; Knödler, A.; Záliš, Stanislav

    2002-01-01

    Roč. 337, - (2002), s. 163-172 ISSN 0020-1693 R&D Projects: GA MŠk OC D14.20 Institutional research plan: CEZ:AV0Z4040901 Keywords : copper compounds * EPR spectroscopy * quinone ligand s Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 1.566, year: 2002

  7. 3D local structure around copper site of rabbit prion-related protein: Quantitative determination by XANES spectroscopy combined with multiple-scattering calculations

    Science.gov (United States)

    Cui, P. X.; Lian, F. L.; Wang, Y.; Wen, Yi; Chu, W. S.; Zhao, H. F.; Zhang, S.; Li, J.; Lin, D. H.; Wu, Z. Y.

    2014-02-01

    Prion-related protein (PrP), a cell-surface copper-binding glycoprotein, is considered to be responsible for a number of transmissible spongiform encephalopathies (TSEs). The structural conversion of PrP from the normal cellular isoform (PrPC) to the post-translationally modified form (PrPSc) is thought to be relevant to Cu2+ binding to histidine residues. Rabbits are one of the few mammalian species that appear to be resistant to TSEs, because of the structural characteristics of the rabbit prion protein (RaPrPC) itself. Here we determined the three-dimensional local structure around the C-terminal high-affinity copper-binding sites using X-ray absorption near-edge structure combined with ab initio calculations in the framework of the multiple-scattering (MS) theory. Result shows that two amino acid resides, Gln97 and Met108, and two histidine residues, His95 and His110, are involved in binding this copper(II) ion. It might help us understand the roles of copper in prion conformation conversions, and the molecular mechanisms of prion-involved diseases.

  8. Morphological development and cytochrome c oxidase activity in Streptomyces lividans are dependent on the action of a copper bound Sco protein

    Science.gov (United States)

    Blundell, Katie L. I. M.; Wilson, Michael T.; Svistunenko, Dimitri A.; Vijgenboom, Erik; Worrall, Jonathan A. R.

    2013-01-01

    Copper has an important role in the life cycle of many streptomycetes, stimulating the developmental switch between vegetative mycelium and aerial hyphae concomitant with the production of antibiotics. In streptomycetes, a gene encoding for a putative Sco-like protein has been identified and is part of an operon that contains two other genes predicted to handle cellular copper. We report on the Sco-like protein from Streptomyces lividans (ScoSl) and present a series of experiments that firmly establish a role for ScoSl as a copper metallochaperone as opposed to a role as a thiol-disulphide reductase that has been assigned to other bacterial Sco proteins. Under low copper concentrations, a Δsco mutant in S. lividans displays two phenotypes; the development switch between vegetative mycelium and aerial hyphae stalls and cytochrome c oxidase (CcO) activity is significantly decreased. At elevated copper levels, the development and CcO activity in the Δsco mutant are restored to wild-type levels and are thus independent of ScoSl. A CcO knockout reveals that morphological development is independent of CcO activity leading us to suggest that ScoSl has at least two targets in S. lividans. We establish that one ScoSl target is the dinuclear CuA domain of CcO and it is the cupric form of ScoSl that is functionally active. The mechanism of cupric ion capture by ScoSl has been investigated, and an important role for a conserved His residue is identified. PMID:23345541

  9. Cloning, crystallization and preliminary X-ray studies of XC2981 from Xanthomonas campestris, a putative CutA1 protein involved in copper-ion homeostasis

    International Nuclear Information System (INIS)

    Lin, Chien-Hung; Chin, Ko-Hsin; Gao, Fei Philip; Lyu, Ping-Chiang; Shr, Hui-Lin; Wang, Andrew H.-J.; Chou, Shan-Ho

    2006-01-01

    A probable copper-ion tolerance protein from the plant pathogen X. campestris has been overexpressed in E. coli, purified and crystallized. Divalent metal ions play key roles in all living organisms, serving as cofactors for many proteins involved in a variety of electron-transfer activities. However, copper ions are highly toxic when an excessive amount is accumulated in a cell. CutA1 is a protein found in all kingdoms of life that is believed to participate in copper-ion tolerance in Escherichia coli, although its specific function remains unknown. Several crystal structures of multimeric CutA1 with different rotation angles and degrees of interaction between trimer interfaces have been reported. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC2981, a possible CutA1 protein present in the plant pathogen Xanthomonas campestris, are reported. The XC2981 crystals diffracted to a resolution of 2.6 Å. They are cubic and belong to space group I23, with unit-cell parameters a = b = c = 130.73 Å

  10. Probing the Composition, Assembly and Activity of Protein Molecular Machines using Native Mass Spectrometry

    NARCIS (Netherlands)

    van de Waterbeemd, M.J.

    2017-01-01

    Native mass spectrometry and mass spectrometry in general, are powerful analytical tools for studying proteins and protein complexes. Native mass spectrometry may provide accurate mass measurements of large macromolecular assemblies enabling the investigation of their composition and stoichiometry.

  11. Probing protein adsorption modes onto poly(ethylene glycol) brushes by neutron reflection

    OpenAIRE

    Schollier, Audrey

    2011-01-01

    Adsorption of proteins at interfaces has an important role in biotechnological and pharmaceutical applications. Indeed, several undesirable processes are related to protein adsorption, as for example: fouling of contact lenses, clotting on blood contacting devices, triggering inflammation around artificial organs, diminished circulation time of therapeutic proteins and drug bearing liposomes. Neutral water soluble polymers, such as poly(ethylene glycol) (PEG), are used to repress protein adso...

  12. Proximity probing assays for simultaneous visualization of protein complexes in situ

    DEFF Research Database (Denmark)

    Moreira, José; Thorsen, Stine Buch; Brünner, Nils

    2013-01-01

    for understanding the biological role(s) of protein complexes and may open new opportunities to improve clinical biomarker research. Leuchowius et al. describe an improved proximity ligation assay for in situ detection of protein complexes, which is able to detect and quantify several protein complexes...

  13. Peptide microarrays to probe for competition for binding sites in a protein interaction network

    NARCIS (Netherlands)

    Sinzinger, M.D.S.; Ruttekolk, I.R.R.; Gloerich, J.; Wessels, H.; Chung, Y.D.; Adjobo-Hermans, M.J.W.; Brock, R.E.

    2013-01-01

    Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins

  14. Removal of copper(II) using deacetylated konjac glucomannan conjugated soy protein isolate.

    Science.gov (United States)

    Liu, Feng; Zou, Hailiang; Peng, Jianbing; Hu, Jinwen; Liu, Hongbo; Chen, Yanwu; Lu, Fenghui

    2016-05-01

    In this study, an environmentally friendly biosorbent deacetylated konjac glucomannan conjugated soy protein isolate (abbreviated as DKGM-C-SPI) was prepared for Cu(2+) ions removal from aqueous solution. Scanning electron microscopy, Fourier transform infrared spectroscopy and zeta potential analysis revealed successful conjugation of soy protein isolate (SPI) onto deacetylated konjac glucomannan (DKGM) matrix. A comparative adsorption performance of DKGM-C-SPI and DKGM was tested to remove Cu(2+) ions from aqueous solution. DKGM-C-SPI showed the desired adsorption performance for Cu(2+) ions. The adsorption equilibrium of DKGM-C-SPI was achieved within 30 min. The adsorption behavior of DKGM-C-SPI followed a pseudo-second-order reaction model. The maximum Cu(2+) ion adsorption capacities obtained from the Langmuir isotherms fit were shown to be 62.50 mg g(-1) for DKGM-C-SPI and 12.23 mg g(-1) for DKGM. This impressive increase about 5 times in Cu(2+) ion adsorption capacity is attributed to the strong Cu(2+) ion chelating ability of the soy protein isolate (SPI) on the DKGM matrix. These results confirm that the DKGM-C-SPI biosorbent has a potential for Cu(2+) ion extraction from wastewater. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Study of Binding between Protein A and Immunoglobulin G Using a Surface Tension Probe

    OpenAIRE

    Yang, L.; Biswas, M. E.; Chen, P.

    2003-01-01

    Molecular interactions and binding are one of the most important and fundamental properties in the study of biochemical and biomedical systems. The understanding of such interactions and binding among biomolecules forms the basis for the design and processing of many biotechnological applications, such as bioseparation and immunoadsorption. In this study, we present a novel method to probe molecular interactions and binding based on surface tension measurement. This method complements convent...

  16. Copper modulates zinc metalloproteinase-dependent ectodomain shedding of key signaling and adhesion proteins and promotes the invasion of prostate cancer epithelial cells

    OpenAIRE

    Parr-Sturgess, Catherine A.; Tinker, Claire L.; Hart, Claire A.; Brown, Michael D.; Clarke, Noel W.; Parkin, Edward T.

    2012-01-01

    A disintegrin and metalloproteinases (ADAMs) and matrix metalloproteinases (MMPs) are zinc metalloproteinases (ZMPs) that catalyze the "ectodomain shedding" of a range of cell surface proteins including signaling and adhesion molecules. These "sheddases" are associated with the invasion and metastasis of a range of cancers. Increased serum and tumor tissue levels of copper are also observed in several cancers, although little is known about how the metal might promote disease progression at t...

  17. New role for an old probe: affinity labeling of oxylipid protein conjugates by N'-aminooxymethylcarbonylhydrazino d-biotin.

    Science.gov (United States)

    Chavez, Juan; Wu, Jianyong; Han, Bingnan; Chung, Woon-Gye; Maier, Claudia S

    2006-10-01

    Free radicals, electrophiles, and endogenous reactive intermediates are generated during normal physiological processes and are capable of modifying DNA, lipids, and proteins. However, elevated levels of oxidative modifications of proteins by reactive species are implicated in the etiology and pathology of oxidative stress-mediated diseases, neurodegeneration, and aging. A mass spectrometry-based approach is reported that aids to the identification and characterization of carbonyl-modified proteins. The method uses N'-aminooxymethylcarbonylhydrazino d-biotin, a biotinylated hydroxylamine derivative that forms an oxime derivative with the aldehyde/keto group found in oxidatively modified proteins. In this paper, the method is demonstrated for one class of carbonyl-modified proteins, namely, oxylipid peptide and protein conjugates formed by Michael addition-type conjugation reactions of alpha,beta-unsaturated aldehydic lipid peroxidation products with nucleophilic peptide side chains. This new application of an "old" probe, which has been used for the detection of abasic sites in DNA strands, introduces a biotin moiety into the oxylipid peptide conjugate. The biotin-modified oxylipid peptide conjugate is then amenable to enrichment using avidin affinity capture. The described method represents an attractive alternative to hydrazine-based derivatization methods for oxidized peptides and proteins because the reduction step necessary for the transformation of the hydrazone bond to the chemically more stable hydrazine bond can be omitted. Tandem mass spectrometry of the labeled oxylipid peptide conjugates indicates that the biotin moiety is at least partially retained on the fragment ion during the collisionally induced dissociation experiments, a prerequisite for the use of automated database searching of uninterpreted tandem mass spectra. The reported approach is outlined for the detection, identification, and characterization of oxylipid peptide conjugates, but the

  18. Probing the Binding Interfaces of Protein Complexes Using Gas-Phase H/D Exchange Mass Spectrometry

    DEFF Research Database (Denmark)

    Mistarz, Ulrik H; Brown, Jeffery M; Haselmann, Kim F

    2016-01-01

    Fast gas-phase hydrogen/deuterium exchange mediated by ND3 gas and measured by mass spectrometry (gas-phase HDX-MS) is a largely unharnessed, fast, and sensitive method for probing primary- and higher-order polypeptide structure. Labeling of heteroatom-bound non-amide hydrogens in a sub...... conditions. Lysozyme ions bound by an oligosaccharide incorporated less deuterium than the unbound ion. Similarly, trypsin ions showed reduced deuterium uptake when bound by the peptide ligand vasopressin. Our results are in good agreement with crystal structures of the native protein complexes......, and illustrate that gas-phase HDX-MS can provide a sensitive and simple approach to measure the number of heteroatom-bound non-amide side-chain hydrogens involved in the binding interface of biologically relevant protein complexes....

  19. Using magnetic nanoparticles to probe protein damage in ferritin caused by freeze concentration

    Directory of Open Access Journals (Sweden)

    E. F. Chagas

    2015-11-01

    Full Text Available We demonstrate a method for monitoring the damage caused to a protein during freeze-thawing in the presence of glycerol, a cryo-protectant. For this work we synthesized magnetite nanoparticles doped with 2.5% cobalt inside the protein ferritin (CMF, dissolved them in different concentration glycerol solutions and measured their magnetization after freezing in a high applied field (5 T. As the temperature was raised, a step-like decrease in the sample magnetization was observed, corresponding to the onset of Brownian relaxation as the viscosity of the freeze-concentrated glycerol solution decreased. The position of the step reveals changes to the protein hydrodynamic radius that we attribute to protein unfolding, while its height depends on how much protein is trapped by ice during freeze concentration. Changes to the protein hydrodynamic radius are confirmed by dynamic light scattering (DLS measurements, but unlike DLS, the magnetic measurements can provide hydrodynamic data while the solution remains mainly frozen.

  20. RGS-Insensitive G Proteins as In Vivo Probes of RGS Function.

    Science.gov (United States)

    Neubig, Richard R

    2015-01-01

    Guanine nucleotide-binding proteins of the inhibitory (Gi/o) class play critical physiological roles and the receptors that activate them are important therapeutic targets (e.g., mu opioid, serotonin 5HT1a, etc.). Gi/o proteins are negatively regulated by regulator of G protein signaling (RGS) proteins. The redundant actions of the 20 different RGS family members have made it difficult to establish their overall physiological role. A unique G protein mutation (G184S in Gαi/o) prevents RGS binding to the Gα subunit and blocks all RGS action at that particular Gα subunit. The robust phenotypes of mice expressing these RGS-insensitive (RGSi) mutant G proteins illustrate the profound action of RGS proteins in cardiovascular, metabolic, and central nervous system functions. Specifically, the enhanced Gαi2 signaling through the RGSi Gαi2(G184S) mutant knock-in mice shows protection against cardiac ischemia/reperfusion injury and potentiation of serotonin-mediated antidepressant actions. In contrast, the RGSi Gαo mutant knock-in produces enhanced mu-opioid receptor-mediated analgesia but also a seizure phenotype. These genetic models provide novel insights into potential therapeutic strategies related to RGS protein inhibitors and/or G protein subtype-biased agonists at particular GPCRs. © 2015 Elsevier Inc. All rights reserved.

  1. Probing structural changes of proteins incorporated into water-in-oil emulsions

    DEFF Research Database (Denmark)

    Jorgensen, Lene; van de Weert, Marco; Vermehren, Charlotte

    2004-01-01

    (BSA) and human serum albumin (HSA) as model proteins. FTIR shows that the overall secondary structure of the proteins changes to some extent, 12% for BSA and 9% for HSA, when these are incorporated into the emulsion. There was no evidence of changes in the distribution of secondary structural elements...... indicated that the domains in the proteins change because the shape of the transition peaks changes, when the proteins were incorporated into the emulsions. The total enthalpy decreases for BSA and HSA when these are incorporated into the emulsion, and some changes to the transition temperatures...

  2. Synthesis and Structure of a Ternary Copper(II) Complex with Mixed Ligands of Diethylenetriamine and Picrate: DNA/Protein-Binding Property and In Vitro Anticancer Activity Studies.

    Science.gov (United States)

    Shi, Ya-Ning; Zheng, Kang; Zhu, Ling; Li, Yan-Tuan; Wu, Zhi-Yong; Yan, Cui-Wei

    2015-05-01

    Based on the importance of the design and synthesis of transition metal complexes with noncovalent DNA/protein-binding abilities in the field of metallo pharmaceuticals, a new mononuclear ternary copper(II) complex with mixed ligands of diethylenetriamine (dien) and picrate anion (pic), identified as [Cu(dien)(pic)](pic), was synthesized and characterized by elemental analysis, molar conductivity measurement, infrared spectrum, electronic spectral studies, and single-crystal X-ray diffractometry. The structure analysis reveals that the copper(II) complex crystallizes in the monoclinic space group P21 /c, and the copper(II) ion has a distorted square pyramidal coordination geometry. A two-dimensional supramolecular structure is formed through hydrogen bonds. The DNA/bovine serum albumin (BSA)-binding properties of the complex are explored, indicating that the complex can interact with herring sperm DNA via intercalation mode and bind to BSA responsible for quenching of tryptophan fluorescence by static quenching mechanism. The in vitro anticancer activity shows that the copper(II) complex is active against the selected tumor cell lines. © 2015 Wiley Periodicals, Inc.

  3. Probing protein interactions with hydrogen/deuterium exchange and mass spectrometry—A review

    International Nuclear Information System (INIS)

    Percy, Andrew J.; Rey, Martial; Burns, Kyle M.; Schriemer, David C.

    2012-01-01

    Highlights: ► Protein chemistry generates mass shifts useful for structure–function studies. ► H/DX supports a powerful mass shift method for protein interaction analysis. ► H/DX mass shifts are useful for determining binding data (K d , off-rates). ► Improved H/DX–MS workflows can accommodate complex protein systems. - Abstract: Assessing the functional outcome of protein interactions in structural terms is a goal of structural biology, however most techniques have a limited capacity for making structure–function determinations with both high resolution and high throughput. Mass spectrometry can be applied as a reader of protein chemistries in order to fill this void, and enable methodologies whereby protein structure–function determinations may be made on a proteome-wide level. Protein hydrogen/deuterium exchange (H/DX) offers a chemical labeling strategy suitable for tracking changes in “dynamic topography” and thus represents a powerful means of monitoring protein structure–function relationships. This review presents the exchange method in the context of interaction analysis. Applications involving interface detection, quantitation of binding, and conformational responses to ligation are discussed, and commentary on recent analytical developments is provided.

  4. Electrostatic interactions in protein adsorption probed by comparing lysozyme and succinylated lysozyme

    NARCIS (Netherlands)

    Veen, van der M.; Norde, W.; Cohen Stuart, M.A.

    2004-01-01

    The influence of electrostatic interactions on protein adsorption was studied by comparing the adsorption of lysozyme and succinylated lysozyme at silica surfaces. The succinylation affects the charge of the protein, but also the stability. Although changes in stability can have an influence on

  5. Performance characteristics, plasma lipids and copper residue in ...

    African Journals Online (AJOL)

    Copper proteinate) and inorganic (Copper sulphate) Cu source on growth performance, plasma lipids and copper residue in organs and tissues of cockerel chickens. 240 day-old commercial Black-Harco cockerel chicks were randomly distributed to ...

  6. Protein Structure-Function Correlation in Living Human Red Blood Cells Probed by Isotope Exchange-based Mass Spectrometry.

    Science.gov (United States)

    Narayanan, Sreekala; Mitra, Gopa; Muralidharan, Monita; Mathew, Boby; Mandal, Amit K

    2015-12-01

    To gain insight into the underlying mechanisms of various biological events, it is important to study the structure-function correlation of proteins within cells. Structural probes used in spectroscopic tools to investigate protein conformation are similar across all proteins. Therefore, structural studies are restricted to purified proteins in vitro and these findings are extrapolated in cells to correlate their functions in vivo. However, due to cellular complexity, in vivo and in vitro environments are radically different. Here, we show a novel way to monitor the structural transition of human hemoglobin upon oxygen binding in living red blood cells (RBCs), using hydrogen/deuterium exchange-based mass spectrometry (H/DX-MS). Exploiting permeability of D2O across cell membrane, the isotope exchange of polypeptide backbone amide hydrogens of hemoglobin was carried out inside RBCs and monitored using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). To explore the conformational transition associated with oxygenation of hemoglobin in vivo, the isotope exchange kinetics was simplified using the method of initial rates. RBC might be considered as an in vivo system of pure hemoglobin. Thus, as a proof-of-concept, the observed results were correlated with structural transition of hemoglobin associated with its function established in vitro. This is the first report on structural changes of a protein upon ligand binding in its endogenous environment. The proposed method might be applicable to proteins in their native state, irrespective of location, concentration, and size. The present in-cell approach opens a new avenue to unravel a plethora of biological processes like ligand binding, folding, and post-translational modification of proteins in living cells.

  7. Hybrid method to solve HP model on 3D lattice and to probe protein stability upon amino acid mutations.

    Science.gov (United States)

    Guo, Yuzhen; Tao, Fengying; Wu, Zikai; Wang, Yong

    2017-09-21

    Predicting protein structure from amino acid sequence is a prominent problem in computational biology. The long range interactions (or non-local interactions) are known as the main source of complexity for protein folding and dynamics and play the dominant role in the compact architecture. Some simple but exact model, such as HP model, captures the pain point for this difficult problem and has important implications to understand the mapping between protein sequence and structure. In this paper, we formulate the biological problem into optimization model to study the hydrophobic-hydrophilic model on 3D square lattice. This is a combinatorial optimization problem and known as NP-hard. Particle swarm optimization is utilized as the heuristic framework to solve the hard problem. To avoid premature in computation, we incorporated the Tabu search strategy. In addition, a pulling strategy was designed to accelerate the convergence of algorithm based on the characteristic of native protein structure. Together a novel hybrid method combining particle swarm optimization, Tabu strategy, and pulling strategy can fold the amino acid sequences on 3D square lattice efficiently. Promising results are reported in several examples by comparing with existing methods. This allows us to use this tool to study the protein stability upon amino acid mutation on 3D lattice. In particular, we evaluate the effect of single amino acid mutation and double amino acids mutation via 3D HP lattice model and some useful insights are derived. We propose a novel hybrid method to combine several heuristic strategies to study HP model on 3D lattice. The results indicate that our hybrid method can predict protein structure more accurately and efficiently. Furthermore, it serves as a useful tools to probe the protein stability on 3D lattice and provides some biological insights.

  8. Essential role of copper in the activity and regular periodicity of a recombinant, tumor-associated, cell surface, growth-related and time-keeping hydroquinone (NADH) oxidase with protein disulfide-thiol interchange activity (ENOX2).

    Science.gov (United States)

    Tang, Xiaoyu; Chueh, P-J; Jiang, Ziying; Layman, Sara; Martin, Berdine; Kim, Chinpal; Morré, Dorothy M; Morré, D James

    2010-10-01

    ECTO-NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit time-keeping and prion-like properties. A bacterially expressed truncated recombinant 46 kDa ENOX2 with full ENOX2 activity bound ca 2 moles copper and 2 moles of zinc per mole of protein. Unfolding of the protein in trifluoroacetic acid in the presence of the copper chelator bathocuproine resulted in reversible loss of both enzymatic activities and of a characteristic pattern in the Amide I to Amide II ratios determined by FTIR with restoration by added copper. The H546-V-H together with His 562 form one copper binding site and H582 represents a second copper site as determined from site-directed mutagenesis. Bound copper emerges as having an essential role in ENOX2 both for enzymatic activity and for the structural changes that underly the periodic alternations in activity that define the time-keeping cycle of the protein.

  9. Probing the binding between norbixin and dairy proteins by spectroscopy methods.

    Science.gov (United States)

    Zhang, Yue; Zhong, Qixin

    2013-08-15

    Annatto (norbixin) has been used to color cheeses for centuries, but there is very little knowledge about interactions between the pigment and dairy proteins. In this study, binding of norbixin with whey protein isolate (WPI), sodium caseinate (NaCN), and 6 individual dairy proteins was investigated by using fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD) and differential scanning calorimetry (DSC). Norbixin was observed to effectively quench the fluorescence of WPI and NaCN by forming complexes. The binding affinity between NaCN and norbixin was higher than that of WPI-norbixin. For individual proteins, bovine serum albumin had higher binding affinity with norbixin than β-lactoglobulin and α-lactalbumin, while κ-casein bound with norbixin better than α- and β-caseins. Binding changed the conformation of WPI and NaCN, but the extent and trend varied for individual proteins. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes

    Directory of Open Access Journals (Sweden)

    Shingo Sotoma

    2016-03-01

    Full Text Available The impeccable photostability of fluorescent nanodiamonds (FNDs is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface.

  11. In Situ STM and AFM of the Copper Protein Pseudomonas Aeruginosa Azurin

    DEFF Research Database (Denmark)

    Friis, Esben P.; Andersen, Jens Enevold Thaulov; Madsen, L.L.

    1997-01-01

    Scanning tunnel (STM) and atomic force microscopy (AFM) in the in situ mode under potentiostatic control have opened new perspectives for mapping the two-dimensional organization of surface adsorbates in aqueous solution. In situ STM and AFM, however, also raise recognized problems. In the context...... exponentially with increasing distance with a decay constant of 0.4–0.5 Å−1. In comparison in situ AFM shows structures laterally convoluted with the tip while the vertical extension is in the same range as the structural size of azurin. The results are of interest in relation to electron tunnel mechanisms...... of redox metalloproteins and in technological contexts such as electrochemical biosensors, microbial corrosion and broadly for protein adsorption from biological liquids....

  12. Development of a novel fluorescent imaging probe for tumor hypoxia by use of a fusion protein with oxygen-dependent degradation domain of HIF-1α

    Science.gov (United States)

    Tanaka, Shotaro; Kizaka-Kondoh, Shinae; Harada, Hiroshi; Hiraoka, Masahiro

    2007-02-01

    More malignant tumors contain more hypoxic regions. In hypoxic tumor cells, expression of a series of hypoxiaresponsive genes related to malignant phenotype such as angiogenesis and metastasis are induced. Hypoxia-inducible factor-1 (HIF-1) is a master transcriptional activator of such genes, and thus imaging of hypoxic tumor cells where HIF-1 is active, is important in cancer therapy. We have been developing PTD-ODD fusion proteins, which contain protein transduction domain (PTD) and the VHL-mediated protein destruction motif in oxygen-dependent degradation (ODD) domain of HIF-1 alpha subunit (HIF-1α). Thus PTD-ODD fusion proteins can be delivered to any tissue in vivo through PTD function and specifically stabilized in hypoxic cells through ODD function. To investigate if PTD-ODD fusion protein can be applied to construct hypoxia-specific imaging probes, we first constructed a fluorescent probe because optical imaging enable us to evaluate a probe easily, quickly and economically in a small animal. We first construct a model fusion porein PTD-ODD-EGFP-Cy5.5 named POEC, which is PTD-ODD protein fused with EGFP for in vitro imaging and stabilization of fusion protein, and conjugated with a near-infrared dye Cy5.5. This probe is designed to be degraded in normoxic cells through the function of ODD domain and followed by quick clearance of free fluorescent dye. On the other hand, this prove is stabilized in hypoxic tumor cells and thus the dye is stayed in the cells. Between normoxic and hypoxic conditions, the difference in the clearance rate of the dye will reveals suited contrast for tumor-hypoxia imaging. The optical imaging probe has not been optimized yet but the results presented here exhibit a potential of PTD-ODD fusion protein as a hypoxia-specific imaging probe.

  13. Detection of copper ions in drinking water using the competitive adsorption of proteins.

    Science.gov (United States)

    Wang, Ran; Wang, Wei; Ren, Hao; Chae, Junseok

    2014-07-15

    Heavy metal ions, i.e., Cu(2+), are harmful to the environment and our health. In order to detect them, and circumvent or alleviate the weaknesses of existing detecting technologies, we contrive a unique Surface Plasmon Resonance (SPR) biosensor combined with competitive adsorption of proteins, termed the Vroman effect. This approach adopts native proteins (albumin) as bio-receptors that interact with Cu(2+) to be denatured. Denaturation disrupts the conformation of albumin so that it weakens its affinity to adsorb on the sensing surface. Through the competitive adsorption between the denatured albumins and the native ones, the displacement occurs adjacent to the sensing surface, and this process is real-time monitored by SPR, a surface-sensitive label-free biosensor. The affinities of native albumin is significantly higher than that of denatured albumin, demonstrated by measured KD of native and denatured albumin to gold surafce, 5.8±0.2×10(-5) M and 5.4±0.1×10(-4) M, respectively. Using our biosensor, Cu(2+) with concentration down to 0.1mg/L is detected in PBS, tap water, deionized water, and bottled water. The SPR biosensor is characterized for 5 different heavy metal ions, Cu(2+), Fe(3+), Mn(2+), Pb(2+), and Hg(2+), most common heavy metal ions found in tap water. At the maximum contaminant level (MCL) suggested by the United States Environmental Protection Agency (EPA), the SPR biosensor produces 13.5±0.4, 1.5±0.4, 0, 0, and 0 mDeg, respectively, suggesting the biosensor may be used to detect Cu(2+) in tap water samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. 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.

  15. The N-B Interaction through a Water Bridge: Understanding the Chemoselectivity of a Fluorescent Protein Based Probe for Peroxynitrite.

    Science.gov (United States)

    Chen, Zhi-jie; Tian, Ziqi; Kallio, Karen; Oleson, April L; Ji, Ao; Borchardt, Dan; Jiang, De-en; Remington, S James; Ai, Hui-wang

    2016-04-13

    Boronic acid and esters have been extensively utilized for molecular recognition and chemical sensing. We recently reported a genetically encoded peroxynitrite (ONOO(-))-specific fluorescent sensor, pnGFP, based on the incorporation of a boronic acid moiety into a circularly permuted green fluorescent protein (cpGFP) followed by directed protein evolution. Different from typical arylboronic acids and esters, the chromophore of pnGFP is unreactive to millimolar concentrations of hydrogen peroxide (H2O2). The focus of this study is to explore the mechanism for the observed unusual chemoselectivity of pnGFP toward peroxynitrite over hydrogen peroxide by using site-directed mutagenesis, X-ray crystallography, (11)B NMR, and computational analysis. Our data collectively support that a His residue on the protein scaffold polarizes a water molecule to induce the formation of an sp(3)-hybridized boron in the chromophore, thereby tuning the reactivity of pnGFP with various reactive oxygen and nitrogen species (ROS/RNS). Our study demonstrates the first example of tunable boron chemistry in a folded nonnative protein, which offers wide implications in designing selective chemical probes.

  16. Proteomic Analysis of Copper-Binding Proteins in Excess Copper-Stressed Roots of Two Rice (Oryza sativa L. Varieties with Different Cu Tolerances.

    Directory of Open Access Journals (Sweden)

    Chen Chen

    Full Text Available To better understand the mechanisms involved in the heavy metal stress response and tolerance in plants, a proteomic approach was used to investigate the differences in Cu-binding protein expression in Cu-tolerant and Cu-sensitive rice varieties. Cu-binding proteins from Cu-treated rice roots were separated using a new IMAC method in which an IDA-sepharose column was applied prior to the Cu-IMAC column to remove metal ions from protein samples. More than 300 protein spots were reproducibly detected in the 2D gel. Thirty-five protein spots exhibited changes greater than 1.5-fold in intensity compared to the control. Twenty-four proteins contained one or more of nine putative metal-binding motifs reported by Smith et al., and 19 proteins (spots contained one to three of the top six motifs reported by Kung et al. The intensities of seven protein spots were increased in the Cu-tolerant variety B1139 compared to the Cu-sensitive variety B1195 (p<0.05 and six protein spots were markedly up-regulated in B1139, but not detectable in B1195. Four protein spots were significantly up-regulated in B1139, but unchanged in B1195 under Cu stress. In contrast, two protein spots were significantly down-regulated in B1195, but unchanged in B1139. These Cu-responsive proteins included those involved in antioxidant defense and detoxification (spots 5, 16, 21, 22, 28, 29 and 33, pathogenesis (spots 5, 16, 21, 22, 28, 29 and 33, regulation of gene transcription (spots 8 and 34, amino acid synthesis (spots 8 and 34, protein synthesis, modification, transport and degradation (spots 1, 2, 4, 10, 15, 19, 30, 31, 32 and 35, cell wall synthesis (spot 14, molecular signaling (spot 3, and salt stress (spots 7, 9 and 27; together with other proteins, such as a putative glyoxylate induced protein, proteins containing dimeric alpha-beta barrel domains, and adenosine kinase-like proteins. Our results suggest that these proteins, together with related physiological processes, play

  17. Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Hansen, Jakob L; Sheikh, Søren P

    2002-01-01

    The calcium-sensing receptor (CaR) belongs to family C of the G-protein coupled receptor superfamily. The receptor is believed to exist as a homodimer due to covalent and non-covalent interactions between the two amino terminal domains (ATDs). It is well established that agonist binding to family C...... receptors takes place at the ATD and that this causes the ATD dimer to twist. However, very little is known about the translation of the ATD dimer twist into G-protein coupling to the 7 transmembrane moieties (7TMs) of these receptor dimers. In this study we have attempted to delineate the agonist......-induced intermolecular movements in the CaR homodimer using the new bioluminescence resonance energy transfer technique, BRET2, which is based on the transference of energy from Renilla luciferase (Rluc) to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations...

  18. Dual Functional Small Molecule Probes as Fluorophore and Ligand for Misfolding Proteins

    OpenAIRE

    Zhang, Xueli; Ran, Chongzhao

    2013-01-01

    Misfolding of a protein is a destructive process for variety of diseases that include neurodegenerative diseases such as Alzheimer’s disease, Parkinson disease, Huntington disease, mad cow disease, amyotrophic lateral sclerosis (ALS), and frontal temporal dementia (FTD), and other non-CNS diseases such as diabetes, cystic fibrosis, and lysosomal storage diseases. Formation of various misfunctional large assembles of the misfolded protein is the primary consequence. To detect the formation of ...

  19. Disulfiram/copper-disulfiram Damages Multiple Protein Degradation and Turnover Pathways and Cytotoxicity is Enhanced by Metformin in Oesophageal Squamous Cell Carcinoma Cell Lines.

    Science.gov (United States)

    Jivan, Rupal; Damelin, Leonard Howard; Birkhead, Monica; Rousseau, Amanda Louise; Veale, Robin Bruce; Mavri-Damelin, Demetra

    2015-10-01

    Disulfiram (DSF), used since the 1950s in the treatment of alcoholism, is reductively activated to diethyldithiocarbamate and both compounds are thiol-reactive and readily complex copper. More recently DSF and copper-DSF (Cu-DSF) have been found to exhibit potent anticancer activity. We have previously shown that the anti-diabetic drug metformin is anti-proliferative and induces an intracellular reducing environment in oesophageal squamous cell carcinoma (OSCC) cell lines. Based on these observations, we investigated the effects of Cu-DSF and DSF, with and without metformin, in this present study. We found that Cu-DSF and DSF caused considerable cytotoxicity across a panel of OSCC cells, and metformin significantly enhanced the effects of DSF. Elevated copper transport contributes to DSF and metformin-DSF-induced cytotoxicity since the cell-impermeable copper chelator, bathocuproinedisulfonic acid, partially reversed the cytotoxic effects of these drugs, and interestingly, metformin-treated OSCC cells contained higher intracellular copper levels. Furthermore, DSF may target cancer cells preferentially due to their high dependence on protein degradation/turnover pathways, and we found that metformin further enhances the role of DSF as a proteasome inhibitor. We hypothesized that the lysosome could be an additional, novel, target of DSF. Indeed, this acid-labile compound decreased lysosomal acidification, and DSF-metformin co-treatment interfered with the progression of autophagy in these cells. In summary, this is the first such report identifying the lysosome as a target of DSF and based on the considerable cytotoxic effects of DSF either alone or in the presence of metformin, in vitro, and we propose these as novel potential chemotherapeutic approaches for OSCC. © 2015 Wiley Periodicals, Inc.

  20. Mobility Measurements Probe Conformational Changes in Membrane Proteins due to Tension

    Science.gov (United States)

    Morris, Richard G.; Turner, Matthew S.

    2015-11-01

    The function of membrane-embedded proteins such as ion channels depends crucially on their conformation. We demonstrate how conformational changes in asymmetric membrane proteins may be inferred from measurements of their diffusion. Such proteins cause local deformations in the membrane, which induce an extra hydrodynamic drag on the protein. Using membrane tension to control the magnitude of the deformations, and hence the drag, measurements of diffusivity can be used to infer—via an elastic model of the protein—how conformation is changed by tension. Motivated by recent experimental results [Quemeneur et al., Proc. Natl. Acad. Sci. U.S.A. 111, 5083 (2014)], we focus on KvAP, a voltage-gated potassium channel from Aeropyrum pernix. The conformation of KvAP is found to change considerably due to tension, with its "walls," where the protein meets the membrane, undergoing significant angular strains. The torsional stiffness is determined to be 26.8 kBT per radian at room temperature. This has implications for both the structure and the function of such proteins in the environment of a tension-bearing membrane.

  1. Copper and Zinc Interactions with Cellular Prion Proteins Change Solubility of Full-Length Glycosylated Isoforms and Induce the Occurrence of Heterogeneous Phenotypes.

    Directory of Open Access Journals (Sweden)

    Svetlana Brim

    Full Text Available Prion diseases are characterized biochemically by protein aggregation of infectious prion isoforms (PrPSc, which result from the conformational conversion of physiological prion proteins (PrPC. PrPC are variable post-translationally modified glycoproteins, which exist as full length and as aminoterminally truncated glycosylated proteins and which exhibit differential detergent solubility. This implicates the presence of heterogeneous phenotypes, which overlap as protein complexes at the same molecular masses. Although the biological function of PrPC is still enigmatic, evidence reveals that PrPC exhibits metal-binding properties, which result in structural changes and decreased solubility. In this study, we analyzed the yield of PrPC metal binding affiliated with low solubility and changes in protein banding patterns. By implementing a high-speed centrifugation step, the interaction of zinc ions with PrPC was shown to generate large quantities of proteins with low solubility, consisting mainly of full-length glycosylated PrPC; whereas unglycosylated PrPC remained in the supernatants as well as truncated glycosylated proteins which lack of octarepeat sequence necessary for metal binding. This effect was considerably lower when PrPC interacted with copper ions; the presence of other metals tested exhibited no effect under these conditions. The binding of zinc and copper to PrPC demonstrated differentially soluble protein yields within distinct PrPC subtypes. PrPC-Zn2+-interaction may provide a means to differentiate glycosylated and unglycosylated subtypes and offers detailed analysis of metal-bound and metal-free protein conversion assays.

  2. Copper and Zinc Interactions with Cellular Prion Proteins Change Solubility of Full-Length Glycosylated Isoforms and Induce the Occurrence of Heterogeneous Phenotypes

    Science.gov (United States)

    Brim, Svetlana; Groschup, Martin H.; Kuczius, Thorsten

    2016-01-01

    Prion diseases are characterized biochemically by protein aggregation of infectious prion isoforms (PrPSc), which result from the conformational conversion of physiological prion proteins (PrPC). PrPC are variable post-translationally modified glycoproteins, which exist as full length and as aminoterminally truncated glycosylated proteins and which exhibit differential detergent solubility. This implicates the presence of heterogeneous phenotypes, which overlap as protein complexes at the same molecular masses. Although the biological function of PrPC is still enigmatic, evidence reveals that PrPC exhibits metal-binding properties, which result in structural changes and decreased solubility. In this study, we analyzed the yield of PrPC metal binding affiliated with low solubility and changes in protein banding patterns. By implementing a high-speed centrifugation step, the interaction of zinc ions with PrPC was shown to generate large quantities of proteins with low solubility, consisting mainly of full-length glycosylated PrPC; whereas unglycosylated PrPC remained in the supernatants as well as truncated glycosylated proteins which lack of octarepeat sequence necessary for metal binding. This effect was considerably lower when PrPC interacted with copper ions; the presence of other metals tested exhibited no effect under these conditions. The binding of zinc and copper to PrPC demonstrated differentially soluble protein yields within distinct PrPC subtypes. PrPC–Zn2+-interaction may provide a means to differentiate glycosylated and unglycosylated subtypes and offers detailed analysis of metal-bound and metal-free protein conversion assays. PMID:27093554

  3. Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Hansen, Jakob L; Sheikh, Søren P

    2002-01-01

    -induced intermolecular movements in the CaR homodimer using the new bioluminescence resonance energy transfer technique, BRET2, which is based on the transference of energy from Renilla luciferase (Rluc) to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations...

  4. Nitrate as a probe of cytochrome c surface : crystallographic identification of crucial "hot spots" for protein-protein recognition

    NARCIS (Netherlands)

    De March, Matteo; Demitri, Nicola; De Zorzi, Rita; Casini, Angela; Gabbiani, Chiara; Guerri, Annalisa; Messori, Luigi; Geremia, Silvano

    The electrostatic surface of cytochrome c and its changes with the iron oxidation state are involved in the docking and undocking processes of this protein to its biological partners in the mitochondrial respiratory pathway. To investigate the subtle mechanisms of formation of productive

  5. Atomic force microscopy and spectroscopy to probe single membrane proteins in lipid bilayers.

    Science.gov (United States)

    Sapra, K Tanuj

    2013-01-01

    The atomic force microscope (AFM) has opened vast avenues hitherto inaccessible to the biological scientist. The high temporal (millisecond) and spatial (nanometer) resolutions of the AFM are suited for studying many biological processes in their native conditions. The AFM cantilever stylus is aptly termed as a "lab on a tip" owing to its versatility as an imaging tool as well as a handle to manipulate single bonds and proteins. Recent examples assert that the AFM can be used to study the mechanical properties and monitor processes of single proteins and single cells, thus affording insight into important mechanistic details. This chapter specifically focuses on practical and analytical protocols of single-molecule AFM methodologies related to high-resolution imaging and single-molecule force spectroscopy of membrane proteins. Both these techniques are operator oriented, and require specialized working knowledge of the instrument, theoretical, and practical skills.

  6. Ultrasensitive probing of the protein resistance of PEG surfaces by secondary ion mass spectrometry

    DEFF Research Database (Denmark)

    Kingshott, P.; McArthur, S.; Thissen, H.

    2002-01-01

    The highly sensitive surface analytical techniques X-ray photoelectron spectroscopy (XPS) and time-of-flight static secondary ion mass spectrometry (ToF-SIMS) were used to test the resistance of poly(ethylene glycol) (PEG) coatings towards adsorption of lysozyme (LYS) and fibronectin (FN). PEG...... temperature to maximise the graft density of the PEG chains. XPS showed that the grafted density of PEG chains was slightly higher on the allylamine surface. XPS detected no adsorption of either protein on either PEG coating. ToF-SIMS analysis, on the other hand, found, in the positive ion spectra, minute...... but statistically significant signals assignable to amino acid fragment ions from both proteins adsorbed to the lower density PEG coating and from LYS but not FN on the higher density PEG coating. Negative ion spectra contained relatively more intense protein fragment ion signals for the lower density PEG coating...

  7. Probing membrane protein structure using water polarization transfer solid-state NMR.

    Science.gov (United States)

    Williams, Jonathan K; Hong, Mei

    2014-10-01

    Water plays an essential role in the structure and function of proteins, lipid membranes and other biological macromolecules. Solid-state NMR heteronuclear-detected (1)H polarization transfer from water to biomolecules is a versatile approach for studying water-protein, water-membrane, and water-carbohydrate interactions in biology. We review radiofrequency pulse sequences for measuring water polarization transfer to biomolecules, the mechanisms of polarization transfer, and the application of this method to various biological systems. Three polarization transfer mechanisms, chemical exchange, spin diffusion and NOE, manifest themselves at different temperatures, magic-angle-spinning frequencies, and pulse irradiations. Chemical exchange is ubiquitous in all systems examined so far, and spin diffusion plays the key role in polarization transfer within the macromolecule. Tightly bound water molecules with long residence times are rare in proteins at ambient temperature. The water polarization-transfer technique has been used to study the hydration of microcrystalline proteins, lipid membranes, and plant cell wall polysaccharides, and to derive atomic-resolution details of the kinetics and mechanism of ion conduction in channels and pumps. Using this approach, we have measured the water polarization transfer to the transmembrane domain of the influenza M2 protein to obtain information on the structure of this tetrameric proton channel. At short mixing times, the polarization transfer rates are site-specific and depend on the pH, labile protons, sidechain conformation, as well as the radial position of the residues in this four-helix bundle. Despite the multiple dependences, the initial transfer rates reflect the periodic nature of the residue positions from the water-filled pore, thus this technique provides a way of gleaning secondary structure information, helix tilt angle, and the oligomeric structure of membrane proteins. Copyright © 2014 Elsevier Inc. All

  8. Probing the flexibility of large conformational changes in protein structures through local perturbations.

    Directory of Open Access Journals (Sweden)

    Bosco K Ho

    2009-04-01

    Full Text Available Protein conformational changes and dynamic behavior are fundamental for such processes as catalysis, regulation, and substrate recognition. Although protein dynamics have been successfully explored in computer simulation, there is an intermediate-scale of motions that has proven difficult to simulate - the motion of individual segments or domains that move independently of the body the protein. Here, we introduce a molecular-dynamics perturbation method, the Rotamerically Induced Perturbation (RIP, which can generate large, coherent motions of structural elements in picoseconds by applying large torsional perturbations to individual sidechains. Despite the large-scale motions, secondary structure elements remain intact without the need for applying backbone positional restraints. Owing to its computational efficiency, RIP can be applied to every residue in a protein, producing a global map of deformability. This map is remarkably sparse, with the dominant sites of deformation generally found on the protein surface. The global map can be used to identify loops and helices that are less tightly bound to the protein and thus are likely sites of dynamic modulation that may have important functional consequences. Additionally, they identify individual residues that have the potential to drive large-scale coherent conformational change. Applying RIP to two well-studied proteins, Dihdydrofolate Reductase and Triosephosphate Isomerase, which possess functionally-relevant mobile loops that fluctuate on the microsecond/millisecond timescale, the RIP deformation map identifies and recapitulates the flexibility of these elements. In contrast, the RIP deformation map of alpha-lytic protease, a kinetically stable protein, results in a map with no significant deformations. In the N-terminal domain of HSP90, the RIP deformation map clearly identifies the ligand-binding lid as a highly flexible region capable of large conformational changes. In the Estrogen

  9. The RNA binding site of S8 ribosomal protein of Escherichia coli: Selex and hydroxyl radical probing studies.

    Science.gov (United States)

    Moine, H; Cachia, C; Westhof, E; Ehresmann, B; Ehresmann, C

    1997-03-01

    The RNA binding site of ribosomal protein S8 of Escherichia coli is confined to a small region within the stem of a hairpin in 16S rRNA (nt 588-605/633-651), and thus represents a model system for understanding RNA/protein interaction rules. The S8 binding site on 16S rRNA was suspected to contain noncanonical features difficult to prove with classical genetical or biochemical means. We performed in vitro iterative selection of RNA aptamers that bind S8. For the different aptamers, the interactions with the protein were probed with hydroxyl radicals. Aptamers that were recognized according to the same structural rules as wild-type RNA, but with variations not found in nature, were identified. These aptamers revealed features in the S8 binding site that had been concealed during previous characterizations by the high base conservation throughout evolution. Our data demonstrate that the core structure of the S8 binding site is composed of three interdependent bases (nt 597/641/643), with an essential intervening adenine nucleotide (position 642). The other elements important for the binding site are a base pair (598/640) above the three interdependent bases and a bulged base at position 595, the identity of which is not important. Possible implications on the geometry of the S8 binding site are discussed with the help of a three-dimensional model.

  10. Influence of ultrasound probe treatment time and protease type on functional and physicochemical characteristics of egg white protein hydrolysates.

    Science.gov (United States)

    Stefanovic, Andrea B; Jovanovic, Jelena R; Balanc, Bojana D; Šekuljica, Nataša Ž; Tanaskovic, Sonja M Jakovetic; Dojcinovic, Marina B; Kneževic-Jugovic, Zorica D

    2018-03-05

    The objective of this study was to discover the relationship between the ultrasound probe treatment (UPT) on egg white proteins (EWPs) before EWPs hydrolysis by different proteases, and the functional properties of the obtained hydrolysates. To fulfill this goal, the protein solubility, foaming, and emulsifying properties were studied as a function of the UPT time and then related to the surface characteristics and structural properties. The changes in the hydrolysates microstructures and macromolecular conformation, induced by the UPT, were followed using scanning electron microscope analyzis (SEM) and Fourier transforms infrared spectroscopy (FTIR). The results showed that UPT influenced (P < 0.05) the proteolysis of egg white proteins for all examined treatment times. Alcalase hydrolysates (AHs) and papain hydrolysates (PHs) were found to have a higher solubility, as a consequence of their relatively higher foaming, and emulsifying properties compared to the untreated hydrolysates. The changes in surface hydrophobicity, sulfhydryl content and surface charge of AHs and PHs indicated unfolding of EWPs affected by ultrasound. SEM analyzis showed that UPT destroyed the microstructures of AHs and PHs, while FTIR spectra indicated remarkable changes in the macromolecular conformation of AHs and PHs after UPT. This study revealed that by combining ultrasound pre-hydrolysis treatment under controlled conditions with thoughtful proteases selection, hydrolysates with improved functional properties could be produced, enhancing utilization of EWPs in food products.

  11. GC-MS identification of proteins in wall painting samples: a fast clean-up procedure to remove copper-based pigment interferences.

    Science.gov (United States)

    Gautier, Gwénaëlle; Colombini, Maria Perla

    2007-08-15

    A new approach was explored to purify proteins in a multi-step procedure for the characterisation of proteinaceous materials (casein, animal glue, and egg) in artwork samples by gas chromatography-mass spectrometry. High concentrations of inorganic salts, such as azurite, have been found to impair the determination of protein via amino acid analysis. The effect of varying concentrations of copper-based pigments on the quantification of amino acids was evaluated through the analysis of replica paintings prepared with the three types of proteinaceous materials. Glycine, aspartic and glutamic acids are the amino acids most affected by the presence of copper salts. In the case of high concentration of salts, this interference hampers the correct identification of the proteins. To eliminate the inorganic salts, a C18 pipette tip was used to clean-up the ammonia extracts before the acidic hydrolysis step. The clean-up procedure allows us to prevent the influence of the inorganic salts and thus allows correct protein identification, though the quantitative recovery of proteinaceous material is quite low. The effectiveness of the optimised procedure was evaluated by analysing samples from two Italian wall paintings from the 13th and the 14th centuries. Without the clean-up it would not have been possible to detect the presence of a mixture of egg and animal glue in one case, and that of egg in the other one.

  12. A subnanomolar fluorescent probe for protein kinase CK2 interaction studies

    DEFF Research Database (Denmark)

    Enkvist, Erki; Viht, Kaido; Bischoff, Nils

    2012-01-01

    assay that used thin layer chromatography for the measurement of the rate of phosphorylation of fluorescently labelled peptide 5-TAMRA-RADDSDDDDD. The most potent inhibitor, ARC-1502 (K(i) = 0.5 nM), revealed high selectivity for CK2α in a panel of 140 protein kinases. Labelling of ARC-1502 with Promo...

  13. Probing simultaneously membrane dynamics and protein activity in suspended bilayers in a microfluidic format

    NARCIS (Netherlands)

    Schulze Greiving-Stimberg, Verena Carolin; Bomer, Johan G.; de Boer, Hans L.; van den Berg, Albert; le Gac, Severine

    2014-01-01

    Membrane dynamics affect the structure and function of ion channels, a point that deserves more attention while studying membrane proteins. One important factor in the local lipidic environment of the ion channels, is the membrane fluidity which is directly connected to the free diffusion and

  14. Probing Bio-Nano Interactions between Blood Proteins and Monolayer-Stabilized Graphene Sheets

    DEFF Research Database (Denmark)

    Gan, Shiyu; Zhong, Lijie; Han, Dongxue

    2015-01-01

    Meeting proteins is regarded as the starting event for nanostructures to enter biological systems. Understanding their interactions is thus essential for a newly emerging field, nanomedicine. Chemically converted graphene (CCG) is a wonderful two-dimesional (2D) material for nanomedecine, but its...

  15. Dynamical transition in molecular glasses and proteins observed by spin relaxation of nitroxide spin probes and labels

    Science.gov (United States)

    Golysheva, Elena A.; Shevelev, Georgiy Yu.; Dzuba, Sergei A.

    2017-08-01

    In glassy substances and biological media, dynamical transitions are observed in neutron scattering that manifests itself as deviations of the translational mean-squared displacement, , of hydrogen atoms from harmonic dynamics. In biological media, the deviation occurs at two temperature intervals, at ˜100-150 K and at ˜170-230 K, and it is attributed to the motion of methyl groups in the former case and to the transition from harmonic to anharmonic or diffusive motions in the latter case. In this work, electron spin echo (ESE) spectroscopy—a pulsed version of electron paramagnetic resonance—is applied to study the spin relaxation of nitroxide spin probes and labels introduced in molecular glass former o-terphenyl and in protein lysozyme. The anisotropic contribution to the rate of the two-pulse ESE decay, ΔW, is induced by spin relaxation appearing because of restricted orientational stochastic molecular motion; it is proportional to τc, where is the mean-squared angle of reorientation of the nitroxide molecule around the equilibrium position and τc is the correlation time of reorientation. The ESE time window allows us to study motions with τc τc temperature dependence shows a transition near 240 K, which is in agreement with the literature data on . For spin probes of essentially different size, the obtained data were found to be close, which evidences that motion is cooperative, involving a nanocluster of several neighboring molecules. For the dry lysozyme, the τc values below 260 K were found to linearly depend on the temperature in the same way as it was observed in neutron scattering for . As spin relaxation is influenced only by stochastic motion, the harmonic motions seen in ESE must be overdamped. In the hydrated lysozyme, ESE data show transitions near 130 K for all nitroxides, near 160 K for the probe located in the hydration layer, and near 180 K for the label in the protein interior. For this system, the two latter transitions are not

  16. Expression of a vacuole-localized BURP-domain protein from soybean (SALI3-2 enhances tolerance to cadmium and copper stresses.

    Directory of Open Access Journals (Sweden)

    Yulin Tang

    Full Text Available The plant-specific BURP family proteins play diverse roles in plant development and stress responses, but the function mechanism of these proteins is still poorly understood. Proteins in this family are characterized by a highly conserved BURP domain with four conserved Cys-His repeats and two other Cys, indicating that these proteins potentially interacts with metal ions. In this paper, an immobilized metal affinity chromatography (IMAC assay showed that the soybean BURP protein SALI3-2 could bind soft transition metal ions (Cd(2+, Co(2+, Ni(2+, Zn(2+ and Cu(2+ but not hard metal ions (Ca(2+ and Mg(2+ in vitro. A subcellular localization analysis by confocal laser scanning microscopy revealed that the SALI3-2-GFP fusion protein was localized to the vacuoles. Physiological indexes assay showed that Sali3-2-transgenic Arabidopsis thaliana seedlings were more tolerant to Cu(2+ or Cd(2+ stresses than the wild type. An inductively coupled plasma optical emission spectrometry (ICP-OES analysis illustrated that, compared to the wild type seedlings the Sali3-2-transgenic seedlings accumulated more cadmium or copper in the roots but less in the upper ground tissues when the seedlings were exposed to excessive CuCl2 or CdCl2 stress. Therefore, our findings suggest that the SALI3-2 protein may confer cadmium (Cd(2+ and copper (Cu(2+ tolerance to plants by helping plants to sequester Cd(2+ or Cu(2+ in the root and reduce the amount of heavy metals transported to the shoots.

  17. Probing the Ca2+-assisted pi-pi interaction during Ca2+-dependent protein folding

    Czech Academy of Sciences Publication Activity Database

    Matyska Lišková, Petra; Fišer, Radovan; Macek, Pavel; Chmelík, Josef; Sýkora, Jan; Bednárová, Lucie; Konopásek, I.; Bumba, Ladislav

    2016-01-01

    Roč. 12, č. 2 (2016), s. 531-541 ISSN 1744-683X R&D Projects: GA ČR(CZ) GAP207/11/0717; GA ČR(CZ) GBP208/12/G016; GA MŠk LO1509 Institutional support: RVO:61388971 ; RVO:61388963 ; RVO:61388955 Keywords : METAL-ION-BINDING * NEISSERIA-MENINGITIDIS * RTX PROTEINS Subject RIV: CE - Biochemistry; CF - Physical ; Theoretical Chemistry (UFCH-W) Impact factor: 3.889, year: 2016

  18. Probing heme protein-ligand interactions by UV/visible absorption spectroscopy.

    Science.gov (United States)

    Nienhaus, Karin; Nienhaus, G Ulrich

    2005-01-01

    Ultraviolet/visible (UV/vis) absorption spectroscopy is a powerful tool for steady-state and time-resolved studies of protein-ligand interactions. Prosthetic groups in proteins frequently have strong electronic absorbance bands that depend on the oxidation, ligation, and conformation states of the chromophores. They are also sensitive to conformational changes of the polypeptide chain into which they are embedded. Steady-state absorption spectroscopy provides information on ligand binding equilibria, from which the Gibbs free energy differences between the ligated and unligated states can be computed. Time-resolved absorption spectroscopy allows one to detect short-lived intermediate states that may not get populated significantly under equilibrium conditions, but may nevertheless be of crucial importance for biological function. Moreover, the energy barriers that have to be surmounted in the reaction can be determined. In this chapter, we present a number of typical applications of steady-state and ns time-resolved UV/vis absorption spectroscopy in the study of ligand binding to the central iron in heme proteins.

  19. Exploiting Uniformly13C-Labeled Carbohydrates for Probing Carbohydrate-Protein Interactions by NMR Spectroscopy.

    Science.gov (United States)

    Nestor, Gustav; Anderson, Taigh; Oscarson, Stefan; Gronenborn, Angela M

    2017-05-03

    NMR of a uniformly 13 C-labeled carbohydrate was used to elucidate the atomic details of a sugar-protein complex. The structure of the 13 C-labeled Manα(1-2)Manα(1-2)ManαOMe trisaccharide ligand, when bound to cyanovirin-N (CV-N), was characterized and revealed that in the complex the glycosidic linkage torsion angles between the two reducing-end mannoses are different from the free trisaccharide. Distances within the carbohydrate were employed for conformational analysis, and NOE-based distance mapping between sugar and protein revealed that Manα(1-2)Manα(1-2)ManαOMe is bound more intimately with its two reducing-end mannoses into the domain A binding site of CV-N than with the nonreducing end unit. Taking advantage of the 13 C spectral dispersion of 13 C-labeled carbohydrates in isotope-filtered experiments is a versatile means for a simultaneous mapping of the binding interactions on both, the carbohydrate and the protein.

  20. Presenilin promotes dietary copper uptake.

    Directory of Open Access Journals (Sweden)

    Adam Southon

    Full Text Available Dietary copper is essential for multicellular organisms. Copper is redox active and required as a cofactor for enzymes such as the antioxidant Superoxide Dismutase 1 (SOD1. Copper dyshomeostasis has been implicated in Alzheimer's disease. Mutations in the presenilin genes encoding PS1 and PS2 are major causes of early-onset familial Alzheimer's disease. PS1 and PS2 are required for efficient copper uptake in mammalian systems. Here we demonstrate a conserved role for presenilin in dietary copper uptake in the fly Drosophila melanogaster. Ubiquitous RNA interference-mediated knockdown of the single Drosophila presenilin (PSN gene is lethal. However, PSN knockdown in the midgut produces viable flies. These flies have reduced copper levels and are more tolerant to excess dietary copper. Expression of a copper-responsive EYFP construct was also lower in the midgut of these larvae, indicative of reduced dietary copper uptake. SOD activity was reduced by midgut PSN knockdown, and these flies were sensitive to the superoxide-inducing chemical paraquat. These data support presenilin being needed for dietary copper uptake in the gut and so impacting on SOD activity and tolerance to oxidative stress. These results are consistent with previous studies of mammalian presenilins, supporting a conserved role for these proteins in mediating copper uptake.

  1. Probing the Mutational Interplay between Primary and Promiscuous Protein Functions: A Computational-Experimental Approach

    Science.gov (United States)

    Garcia-Seisdedos, Hector; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2012-01-01

    Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous

  2. Chemical Synthesis of a Glycopeptide Derived from Skp1 for Probing Protein Specific Glycosylation.

    Science.gov (United States)

    Chinoy, Zoeisha S; Schafer, Christopher M; West, Christopher M; Boons, Geert-Jan

    2015-08-10

    Skp1 is a cytoplasmic and nuclear protein, best known as an adaptor of the SCF family of E3-ubiquitin ligases that label proteins for their degradation. Skp1 in Dictyostelium is posttranslationally modified on a specific hydroxyproline (Hyp) residue by a pentasaccharide, which consists of a Fucα1,2-Galβ-1,3-GlcNAcα core, decorated with two α-linked Gal residues. A glycopeptide derived form Skp1 was prepared to characterize the α-galactosyltransferase (AgtA) that mediates the addition of the α-Gal moieties, and to develop antibodies suitable for tracking the trisaccharide isoform of Skp1 in cells. A strategy was developed for the synthesis of the core trisaccharide-Hyp based on the use of 2-naphthylmethyl (Nap) ethers as permanent protecting groups to allow late stage installation of the Hyp moiety. Tuning of glycosyl donor and acceptor reactivities was critical for achieving high yields and anomeric selectivities of glycosylations. The trisaccharide-Hyp moiety was employed for the preparation of the glycopeptide using microwave-assisted solid phase peptide synthesis. Enzyme kinetic studies revealed that trisaccharide-Hyp and trisaccharide-peptide are poorly recognized by AgtA, indicating the importance of context provided by the native Skp1 protein for engagement with the active site. The trisaccharide-peptide was a potent immunogen capable of generating a rabbit antiserum that was highly selective toward the trisaccharide isoform of full-length Skp1. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Probing amyloid protein aggregation with optical superresolution methods: from the test tube to models of disease.

    Science.gov (United States)

    Kaminski, Clemens F; Kaminski Schierle, Gabriele S

    2016-10-01

    The misfolding and self-assembly of intrinsically disordered proteins into insoluble amyloid structures are central to many neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Optical imaging of this self-assembly process in vitro and in cells is revolutionizing our understanding of the molecular mechanisms behind these devastating conditions. In contrast to conventional biophysical methods, optical imaging and, in particular, optical superresolution imaging, permits the dynamic investigation of the molecular self-assembly process in vitro and in cells, at molecular-level resolution. In this article, current state-of-the-art imaging methods are reviewed and discussed in the context of research into neurodegeneration.

  4. In vivo incorporation of copper into the iron-exchangeable and manganese-exchangeable superoxide dismutase from Propionibacterium shermanii. Amino acid sequence and identity of the protein moieties.

    Science.gov (United States)

    Meier, B; Sehn, A P; Schininà, M E; Barra, D

    1994-01-15

    Propionibacterium shermanii, an aerotolerant anaerobe, produces an iron-containing or a manganese-containing superoxide dismutase, depending on the metal supplied in the culture medium [Meier, B., Barra, D., Bossa, F., Calabrese, L. & Rotilio, G. (1982) J. Biol. Chem. 257, 13977-13980]. In this study, we demonstrate in vivo incorporation of copper into an active superoxide-dismutase protein when iron and manganese are absent from the growth medium. Superoxide dismutases containing either iron, manganese or copper were isolated from P. shermanii, their complete amino acid sequences were determined and the identity of their protein moieties was established. The polypeptide chain is made up of 201 amino acid residues, corresponding to a molecular mass of 22.6 kDa. From sedimentation equilibrium experiments, the native protein shows a molecular mass of approximately 86 kDa and therefore consists of four identical subunits. The primary structure was compared with the structure of other Fe-superoxide dismutases and Mn-superoxide dismutases, in particular those possessing a strict metal cofactor specificity.

  5. Probing Conformational Dynamics of Tau Protein by Hydrogen/Deuterium Exchange Mass Spectrometry

    Science.gov (United States)

    Huang, Richard Y.-C.; Iacob, Roxana E.; Sankaranarayanan, Sethu; Yang, Ling; Ahlijanian, Michael; Tao, Li; Tymiak, Adrienne A.; Chen, Guodong

    2018-01-01

    Fibrillization of the microtubule-associated protein tau has been recognized as one of the signature pathologies of the nervous system in Alzheimer's disease, progressive supranuclear palsy, and other tauopathies. The conformational transition of tau in the fibrillization process, tau monomer to soluble aggregates to fibrils in particular, remains unclear. Here we report on the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in combination with other biochemical approaches, including Thioflavin S fluorescence measurements, enzyme-linked immunosorbent assay (ELISA), and Western blotting to understand the heparin-induced tau's fibrillization. HDX-MS studies including anti-tau antibody epitope mapping experiments provided molecular level details of the full-length tau's conformational dynamics and its regional solvent accessibility upon soluble aggregates formation. The results demonstrate that R3 region in the full-length tau's microtubule binding repeat region (MTBR) is stabilized in the aggregation process, leaving both N and C terminal regions to be solvent exposed in the soluble aggregates and fibrils. The findings also illustrate the practical utility of orthogonal analytical methodologies for the characterization of protein higher order structure. [Figure not available: see fulltext.

  6. Probing Early Misfolding Events in Prion Protein Mutants by NMR Spectroscopy

    Directory of Open Access Journals (Sweden)

    Gregor Ilc

    2013-08-01

    Full Text Available The post-translational conversion of the ubiquitously expressed cellular form of the prion protein, PrPC, into its misfolded and pathogenic isoform, known as prion or PrPSc, plays a key role in prion diseases. These maladies are denoted transmissible spongiform encephalopathies (TSEs and affect both humans and animals. A prerequisite for understanding TSEs is unraveling the molecular mechanism leading to the conversion process whereby most α-helical motifs are replaced by β-sheet secondary structures. Importantly, most point mutations linked to inherited prion diseases are clustered in the C-terminal domain region of PrPC and cause spontaneous conversion to PrPSc. Structural studies with PrP variants promise new clues regarding the proposed conversion mechanism and may help identify “hot spots” in PrPC involved in the pathogenic conversion. These investigations may also shed light on the early structural rearrangements occurring in some PrPC epitopes thought to be involved in modulating prion susceptibility. Here we present a detailed overview of our solution-state NMR studies on human prion protein carrying different pathological point mutations and the implications that such findings may have for the future of prion research.

  7. Thermodiffusion as a probe of protein hydration for streptavidin and the streptavidin-biotin complex

    Science.gov (United States)

    Niether, Doreen; Sarter, Mona; König, Bernd; Zamponi, Michaela; Fitter, Jörg; Stadler, Andreas; Wiegand, Simone

    2018-01-01

    Molecular recognition via protein-ligand interactions is of fundamental importance to numerous processes in living organisms. Microscale thermophoresis (MST) uses the sensitivity of the thermophoretic response upon ligand binding to access information on the reaction kinetics. Additionally, thermophoresis is promising as a tool to gain information on the hydration layer, as the temperature dependence of the thermodiffusion behaviour is sensitive to solute-solvent interactions. To quantify the influence of structural fluctuations and conformational motion of the protein on the entropy change of its hydration layer upon ligand binding, we combine quasi-elastic incoherent neutron scattering (QENS) and isothermal titration calorimetry (ITC) data from literature. However, preliminary results show that replacing water with deuterated water leads to changes of the thermophoretic measurements, which are similar to the changes observed upon binding by biotin. In order to gain a better understanding of the hydration layer all measurements need to be performed in heavy water. This will open a route to develop a microscopic understanding of the correlation between the strength and number of hydrogen bonds and the thermophoretic behaviour.

  8. High-Resolution Imaging of Proteins in Human Teeth by Scanning Probe Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gruverman, A. [North Carolina State University; Wu, D. [North Carolina State University; Rodriguez, Brian J [ORNL; Kalinin, Sergei V [ORNL; Habelitz, S. [University of California, San Francisco

    2007-01-01

    High-resolution studies of dental tissues are of considerable interest for biomedical engineering and clinical applications. In this paper, we demonstrate the application of piezoresponse force microscopy (PFM) to nanoscale imaging of internal structure of human teeth by monitoring the local mechanical response to an electrical bias applied via a conductive tip. It is shown that PFM is capable of detecting dissimilar components of dental tissues, namely, proteins and calcified matrix, which have resembling morphology but different piezoelectric properties. It is demonstrated that collagen fibrils revealed in chemically treated intertubular dentin exhibit high piezoelectric activity and can be visualized in PFM with spatial resolution of 10 nm. Evidence of the presence of protein inclusions of 100-200 nm wide and several micrometers long in tooth enamel has been obtained. Furthermore, it is found that the peritubular dentin and intertubular dentin exhibit different piezoelectric behavior suggesting different concentration of collagen fibrils. The obtained results demonstrate a high potential of PFM in providing an additional insight into the structure of dental tissues. It is suggested that the PFM approach can be used to study the structure of a wide range of biological materials by monitoring their electromechanical behavior at the nanoscale.

  9. A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations

    Directory of Open Access Journals (Sweden)

    Bahman Delalat

    2016-08-01

    Full Text Available Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response.

  10. Jet-printed copper metallization

    Science.gov (United States)

    Hong, Cheong Min

    Macroelectronics is a technology for making electronic circuits over very large areas at low cost. Flat panel displays, sensor arrays, and thin film solar cells are examples of macroelectronics. Crucial to the success of this new technology is the development of inexpensive electronic processes, materials, and devices. Direct printing techniques, which eliminate processing steps and save device and process materials, are the key to high volume and high throughput manufacturing. Copper metallization has been receiving increasing attention in both microelectronics and macroelectronics. Copper has high conductivity, density, melting point, heat capacity, and thermal conductivity. However, copper is also hard to dry etch. For these reasons we have developed and demonstrated a directly printed copper source/drain metallization technique and applied it to the fabrication of hydrogenated amorphous silicon (a-Si:H) thin film transistors (TFTs). The maximum process temperature of 200°C is compatible with conventional active matrix liquid crystal display (AMLCD) technology. In this dissertation, we show the process of depositing copper films by jet printing. We discuss the preparation and the properties of the copper precursor material used in the jet printing. We survey the conversion process from copper precursor to copper under varying processing conditions. The resulting copper film is probed for its physical, electrical, and mechanical properties. To demonstrate the feasibility of the jet printing technique, we print copper source/drain contacts for a-Si:H TFTs. The photolithography-free TFT fabrication process uses the printed xerographic toner technique developed earlier in this laboratory. We show that functional TFTs can be made with printed copper source and drain contacts. The jet printing of copper contacts represents a further step toward an all-printed thin film transistor technology.

  11. Studies of copper transport in mammalian cells using copper radioisotopes

    Energy Technology Data Exchange (ETDEWEB)

    Camakaris, J.; Voskoboinik, I.; Brooks, H.; Greenough, M. [University of Melbourne, Parkville, VIC (Australia). Department of Genetics; Smith, S. [Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW (Australia). Radiopharmaceuticals Division; Mercer, J. [Deakin University, Clayton, VIC (Australia). Centre of Cellular and Molecular Biology

    1998-12-31

    The trace element copper poses a major problem for all organisms. It is essential as a number of vital enzymes require it. Copper deficiency can lead to neurological disorders, osteoporosis and weakening of arteries. However Cu is also highly toxic and homeostatic mechanisms have evolved to maintain Cu at levels which satisfy requirements but do not cause toxicity. Toxicity is mediated by the oxidative capacity of Cu and its ability to generate toxic free radicals. There are several acquired and inherited diseases due to either Cu toxicity or Cu deficiency. The study of these diseases facilitates identification of genes and proteins involved in copper homeostasis, and this in turn will provide rational therapeutic approaches. Using the copper radioisotopes {sup 64}Cu (t1/2 = 12.8 hr) and {sup 67}Cu (t1/2 = 61 hr) we have developed a number of systems for studying copper transport in mammalian cells. These include investigation of copper uptake, copper efflux and ligand blot assays for Cu-binding proteins. Our studies have focused on Menkes disease which is an inherited and usually lethal copper deficiency disorder in humans. We have demonstrated that the Menkes protein is directly involved as a copper efflux pump in mammalian cells. Using cells overexpressing the Menkes protein we have provided the first biochemical evidence that this functions as a Cu translocating (across the membrane) P-type ATPase (Voskoboinik et al., FEBS Letters, in press). These studies were carried out using purified plasma membrane vesicles. We are now carrying out structure- function studies on this protein using targeted mutations and assaying using the radiocopper vesicle assay. Recently we have commenced studies on the role of amyloid precursor protein (APP) in copper transport and relationship of this to Alzheimers disease

  12. An Introduction to Drug Discovery by Probing Protein-Substrate Interactions Using Saturation Transfer Difference-Nuclear Magnetic Resonance (STD-NMR)

    Science.gov (United States)

    Guegan, Jean-Paul; Daniellou, Richard

    2012-01-01

    NMR spectroscopy is a powerful tool for characterizing and identifying molecules and nowadays is even used to characterize complex systems in biology. In the experiment presented here, students learned how to apply this modern technique to probe interactions between small molecules and proteins. With the use of simple organic synthesis, students…

  13. Molecular recognition of carboxylates in the protein leucine zipper by a multivalent supramolecular ligand: residue-specific, sensitive and label-free probing by UV resonance Raman spectroscopy.

    Science.gov (United States)

    Zakeri, B; Niebling, S; Martinéz, A G; Sokkar, P; Sanchez-Garcia, E; Schmuck, C; Schlücker, S

    2018-01-17

    Ultraviolet resonance Raman (UVRR) spectroscopy is a selective, sensitive and label-free vibrational spectroscopic technique. Here, we demonstrate as proof of concept that UVRR can be used for probing the recognition between a multivalent supramolecular ligand and acidic residues in leucine zipper, an α-helical structural motif of many proteins.

  14. Probing the reaction mechanism of IspH protein by x-ray structure analysis

    KAUST Repository

    Gräwert, Tobias

    2009-12-28

    Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) represent the two central intermediates in the biosynthesis of isoprenoids. The recently discovereddeoxyxylulose 5-phosphate pathway generates a mixture of IPP and DMAPP in its final step by reductive dehydroxylation of 1-hydroxy-2-methyl- 2-butenyl 4-diphosphate. This conversion is catalyzed by IspH protein comprising a central iron-sulfur cluster as electron transfer cofactor in the active site. The five crystal structures of IspH in complex with substrate, converted substrate, products and PPi reported in this article provide unique insights into the mechanism of this enzyme. While IspH protein crystallizes with substrate bound to a [4Fe-4S] cluster, crystals of IspH in complex with IPP, DMAPP or inorganic pyrophosphate feature [3Fe-4S] clusters. The IspH:substrate complex reveals a hairpin conformation of the ligand with the C(1) hydroxyl group coordinated to the unique site in a [4Fe-4S] cluster of aconitase type. The resulting alkoxide complex is coupled to a hydrogen-bonding network, which serves as proton reservoir via a Thr167 proton relay. Prolonged x-ray irradiation leads to cleavage of the C(1)-O bond (initiated by reducing photo electrons). The data suggest a reaction mechanism involving a combination of Lewis-acid activation and proton coupled electron transfer. The resulting allyl radical intermediate can acquire a second electron via the iron-sulfur cluster. The reaction may be terminated by the transfer of a proton from the β-phosphate of the substrate to C(1) (affording DMAPP) or C(3) (affording IPP).

  15. X-ray absorption spectroscopic studies of the blue copper site: Metal and ligand K-edge studies to probe the origin of the EPR hyperfine splitting in plastocyanin

    International Nuclear Information System (INIS)

    Shadle, S.E.; Penner-Hahn, J.E.; Schugar, H.J.; Hedman, B.; Hodgson, K.O.; Solomon, E.I.

    1993-01-01

    X-ray absorption spectra for the oxidized blue copper protein plastocyanin and several Cu(II) model complexes have been measured at both the Cu K-edge and the ligand K-edges (Cl and S) in order to elucidate the source of the small parallel hyperfine splitting in the EPR spectra of blue copper centers. Assignment and analysis of a feature in the Cu K-edge X-ray absorption spectrum at ∼8,987 eV as the Cu 1s → 4p + ligand-to-metal charge-transfer shakedown transition has allowed for quantitation of 4p mixing into the ground-state wave function as reflected in the 1s →3d (+4p) intensity at ∼8,979 eV. The results show that distorted tetrahedral (D 2d )CuCl 4 2- is characterized by z mixing, while plastocyanin has only Cu 4p xy mixing. Thus, the small parallel hyperfine splitting in the EPR spectra of D 2d CuCl 4 2- and of oxidized plastocyanin cannot be explained by 12% 4p z mixing into the 3d x 2 -y 2 orbital as had been previously postulated. Data collected at the Cl K-edge for CuCl 4 2- show that the intensity of the ligand pre-edge feature at ∼2,820 eV reflects the degree of covalency between the metal half-occupied orbital and the ligands. The data show that D 2d CuCl 4 2- is not unusually covalent. The source of the small parallel splitting in the EPR of D 2d CuCl 4 2- is discussed. Experiments at the S K-edge (∼2,470 eV) show that plastocyanin is characterized by a highly covalent Cu-S(cysteine) bond relative to the cupric-thiolate model complex [Cu(tet b)(o-SC 6 H 4 CO 2 )]·H 2 O. The XAS results demonstrate that the small parallel hyperfine splitting in the EPR spectra of blue copper sites reflects the high degree of covalency of the copper-thiolate bond. 34 refs., 12 figs., 3 tabs

  16. Thermodynamic Studies of Cu(I) and Other d10 Metal Ions Binding to Proteins in the Copper Homeostasis Pathway and the Organomercurial Detoxification Pathway

    Science.gov (United States)

    Stevenson, Michael James

    Copper(I) is the predominant oxidation state of this essential metal in living cells due to reducing intracellular conditions. Because of deleterious copper-mediated Fenton chemistry, intracellular copper trafficking pathways involve strict regulation by metallochaperone proteins. Previous studies of the 68-residue metallochaperone, HAH1, have shown that it coordinates Cu(I) with two cysteines for transport from Ctr1 in the cell membrane to ATPases in the Golgi network. Using isothermal titration calorimetry (ITC), and methods to suppress oxidation and disproportionation of Cu(I), the thermodynamics of Cu(I), as well as other metal ions, binding to HAH1 have been accurately quantified. During the course of this study, the Cu(I) binding thermodynamics with the stabilizing ligand hexamethyltrien were determined in order to accurately quantify the Cu(I) binding thermodynamics with proteins, and revealed an unexpected Cu(I) coordination chemistry with this ligand. In addition, HAH1 binding the Cu(I) analogue Ag(I), the abundant cellular metal ion Zn(II), and the thiophilic toxic metal ion Hg(II), have been quantified. The binding thermodynamics of these metal ions were also determined in the presence of glutathione to more accurately model physiological conditions. HAH1 has a high affinity for Cu(I), which is both enthalpically and entropically favorable. It has a substantially lower affinity for Zn(II), which is entropically favored, suggesting that Zn(II) is not able to compete with Cu(I) for HAH1 in vivo. However, HAH1 has an exceptionally high affinity for Hg(II), with its larger thiophilicity, and it will displace Cu(I). Mercury(II) and particularly organomercurial compounds are very toxic, yet proteins from the bacterial mer operon provide resistance to this toxicity. In particular, the organomercurial lyase MerB, whose only known structural homologue is a putative copper metallochaperone, is responsible for cleavage of the carbon-mercury bond of MeHg(II) and

  17. Probing the microenvironmental conditions for induction of superficial zone protein expression.

    Science.gov (United States)

    Mhanna, R; Öztürk, E; Schlink, P; Zenobi-Wong, M

    2013-12-01

    To determine the in vitro conditions which promote expression of superficial zone protein (SZP). Chondrocytes from 6-month-old calves were expanded in monolayer culture and the expression of SZP in alginate bead and monolayer culture was quantified with quantitative real time-polymerase chain reaction (qRT-PCR) and immunostaining. The effect of oxygen tension on SZP expression was determined by qRT-PRC analysis of cells cultured in two dimension (2D) and three dimension (3D) under hypoxic (1% pO2) or normoxic (21% pO2) conditions. Finally, to examine the effect of cyclic tensile strain on expression of SZP in 2D and 3D cultures, chondrocytes encapsulated in alginate beams or seeded on type I collagen coated polydimethylsiloxane (PDMS) chambers were subjected to 5% strain at 1 Hz, 2 h/day for 4 days or 2 h at the fourth day of culture and mRNA levels were quantified. Bovine chondrocytes in monolayer showed a drastic decrease in SZP expression, similar in trend to the commonly reported downregulation of type II collagen (Col2). Chondrocytes embedded in alginate beads for 4 days re-expressed SZP but not Col2. SZP expression was higher under normoxic conditions whereas Col2 was upregulated only in alginate beads under hypoxic conditions. Cyclic mechanical strain showed a tendency to upregulate mRNA levels of SZP. A microenvironment encompassing a soft encapsulation material and 21% oxygen is sufficient for fibroblastic chondrocytes to re-express SZP. These results serve as a guideline for the design of stratified engineered articular cartilage and suggest that microenvironmental cues (oxygen tension level) strongly influence the pattern of SZP expression in vivo. Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  18. Cloning and expression analysis of a blue copperbinding protein ...

    African Journals Online (AJOL)

    Adifferentially expressed fragment EST145 was isolated by suppression subtractive hybridization (SSH) method. Using EST145 as the probe, a blue copper-binding protein gene designated as DvBCB was screened from Dasypyrum villosum cDNA Library. The DvBCB gene was 845 bp in length with an open reading frame ...

  19. Copper hypersensitivity

    DEFF Research Database (Denmark)

    Fage, Simon W; Faurschou, Annesofie; Thyssen, Jacob P

    2014-01-01

    . As a metal, it possesses many of the same qualities as nickel, which is a known strong sensitizer. Cumulative data on subjects with presumed related symptoms and/or suspected exposure showed that a weighted average of 3.8% had a positive patch test reaction to copper. We conclude that copper is a very weak...... hypersensitivity, a database search of PubMed was performed with the following terms: copper, dermatitis, allergic contact dermatitis, contact hypersensitivity, contact sensitization, contact allergy, patch test, dental, IUD, epidemiology, clinical, and experimental. Human exposure to copper is relatively common...

  20. Far-Red Fluorescent Probe for Imaging of Vicinal Dithiol-Containing Proteins in Living Cells Based on a pKa Shift Mechanism.

    Science.gov (United States)

    Zhang, Shengrui; Chen, Guojun; Wang, Yuanyuan; Wang, Qin; Zhong, Yaogang; Yang, Xiao-Feng; Li, Zheng; Li, Hua

    2018-02-20

    Vicinal dithiol-containing proteins (VDPs) play fundamental roles in intracellular redox homeostasis and are responsible for many diseases. In this work, we report a far-red fluorescence turn-on probe MCAs for VDPs exploiting the pK a shift of the imine functionality of the probe. MCAs is composed of a merocyanine Schiff base as the fluorescent reporter and a cyclic 1,3,2-dithiarsenolane as the specific ligand for VDPs. The imine pK a of MCAs is 4.8, and it exists predominantly in the Schiff base (SB) form at physiological pH. Due to the absence of a resonating positive charge, it absorbs at a relatively short wavelength and is essentially nonfluorescent. Upon selective binding to reduced bovine serum albumin (rBSA, selected as the model protein), MCAs was brought from aqueous media to the binding pockets of the protein, causing a large increase in pK a value of MCAs (pK a = 7.1). As a result, an increase in the protonated Schiff base (PSB) form of MCAs was observed at the physiological pH conditions, which in turn leads to a bathochromically shifted chromophore (λ abs = 634 nm) and a significant increase in fluorescence intensity (λ em = 657 nm) simultaneously. Furthermore, molecular dynamics simulations indicate that the salt bridges formed between the iminium in MCAs and the residues D72 and D517 in rBSA resist the dissociation of proton from the probe, thus inducing an increase of the pK a value. The proposed probe shows excellent sensitivity and specificity toward VDPs over other proteins and biologically relevant species and has been successfully applied for imaging of VDPs in living cells. We believe that the present pK a shift switching strategy may facilitate the development of new fluorescent probes that are useful for a wide range of applications.

  1. Comparative genomic analyses of copper transporters and cuproproteomes reveal evolutionary dynamics of copper utilization and its link to oxygen.

    Directory of Open Access Journals (Sweden)

    Perry G Ridge

    2008-01-01

    Full Text Available Copper is an essential trace element in many organisms and is utilized in all domains of life. It is often used as a cofactor of redox proteins, but is also a toxic metal ion. Intracellular copper must be carefully handled to prevent the formation of reactive oxygen species which pose a threat to DNA, lipids, and proteins. In this work, we examined patterns of copper utilization in prokaryotes by analyzing the occurrence of copper transporters and copper-containing proteins. Many organisms, including those that lack copper-dependent proteins, had copper exporters, likely to protect against copper ions that inadvertently enter the cell. We found that copper use is widespread among prokaryotes, but also identified several phyla that lack cuproproteins. This is in contrast to the use of other trace elements, such as selenium, which shows more scattered and reduced usage, yet larger selenoproteomes. Copper transporters had different patterns of occurrence than cuproproteins, suggesting that the pathways of copper utilization and copper detoxification are independent of each other. We present evidence that organisms living in oxygen-rich environments utilize copper, whereas the majority of anaerobic organisms do not. In addition, among copper users, cuproproteomes of aerobic organisms were larger than those of anaerobic organisms. Prokaryotic cuproproteomes were small and dominated by a single protein, cytochrome c oxidase. The data are consistent with the idea that proteins evolved to utilize copper following the oxygenation of the Earth.

  2. Copper (II)

    African Journals Online (AJOL)

    CLEMENT O BEWAJI

    ABSTRACT: A Schiff base was prepared from the reaction of 2 - amino - 3 – methylbutanoic acid and 2, 4 - pentanedione. The reaction of the prepared Schiff base with ethanolic solution of copper (II) chloride formed diaquo bis( N – 2 – amino – 3 - methylbutyl - 2, 4 - pentanedionato) copper (II) complex. The Schiff base is ...

  3. Human Thyroid Cancer-1 (TC-1 is a vertebrate specific oncogenic protein that protects against copper and pro-apoptotic genes in yeast

    Directory of Open Access Journals (Sweden)

    Natalie K. Jones

    2015-07-01

    Full Text Available The human Thyroid Cancer-1 (hTC-1 protein, also known as C8orf4 was initially identified as a gene that was up-regulated in human thyroid cancer. Here we show that hTC-1 is a peptide that prevents the effects of over-expressing Bax in yeast. Analysis of the 106 residues of hTC-1 in available protein databases revealed direct orthologues in jawed-vertebrates, including mammals, frogs, fish and sharks. No TC-1 orthologue was detected in lower organisms, including yeast. Here we show that TC-1 is a general pro-survival peptide since it prevents the growth- and cell death-inducing effects of copper in yeast. Human TC-1 also prevented the deleterious effects that occur due to the over-expression of a number of key pro-apoptotic peptides, including YCA1, YBH3, NUC1, and AIF1. Even though the protective effects were more pronounced with the over-expression of YBH3 and YCA1, hTC-1 could still protect yeast mutants lacking YBH3 and YCA1 from the effects of copper sulfate. This suggests that the protective effects of TC-1 are not limited to specific pathways or processes. Taken together, our results indicate that hTC-1 is a pro-survival protein that retains its function when heterologously expressed in yeast. Thus yeast is a useful model to characterize the potential roles in cell death and survival of cancer related genes.

  4. Mobile Probing and Probes

    DEFF Research Database (Denmark)

    Duvaa, Uffe; Ørngreen, Rikke; Weinkouff, Anne-Gitte

    2012-01-01

    Mobile probing is a method, which has been developed for learning about digital work situations, as an approach to discover new grounds. The method can be used when there is a need to know more about users and their work with certain tasks, but where users at the same time are distributed (in time...... and space). Mobile probing was inspired by the cultural probe method, and was influenced by qualitative interview and inquiry approaches. The method has been used in two subsequent projects, involving school children (young adults at 15-17 years old) and employees (adults) in a consultancy company. Findings...... point to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face...

  5. Mobile Probing and Probes

    DEFF Research Database (Denmark)

    Duvaa, Uffe; Ørngreen, Rikke; Weinkouff Mathiasen, Anne-Gitte

    2013-01-01

    Mobile probing is a method, developed for learning about digital work situations, as an approach to discover new grounds. The method can be used when there is a need to know more about users and their work with certain tasks, but where users at the same time are distributed (in time and space......). Mobile probing was inspired by the cultural probe method, and was influenced by qualitative interview and inquiry approaches. The method has been used in two subsequent projects, involving school children (young adults at 15-17 years old) and employees (adults) in a consultancy company. Findings point...... to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face). The development...

  6. DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

    Science.gov (United States)

    Jameson, Eleanor; Taubert, Martin; Coyotzi, Sara; Chen, Yin; Eyice, Özge; Schäfer, Hendrik; Murrell, J Colin; Neufeld, Josh D; Dumont, Marc G

    2017-01-01

    Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13 C, 18 O, or 15 N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labeled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labeled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing of clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labeling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, metagenomes, or metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labeled microorganisms. Analysis of labeled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allow the use of labeled substrates at ecologically relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies

  7. Subunits of highly Fluorescent Protein R-Phycoerythrin as Probes for Cell Imaging and Single-Molecule Detection

    Energy Technology Data Exchange (ETDEWEB)

    Isailovic, Dragan [Iowa State Univ., Ames, IA (United States)

    2005-01-01

    The purposes of our research were: (1) To characterize subunits of highly fluorescent protein R-Phycoerythrin (R-PE) and check their suitability for single-molecule detection (SMD) and cell imaging, (2) To extend the use of R-PE subunits through design of similar proteins that will be used as probes for microscopy and spectral imaging in a single cell, and (3) To demonstrate a high-throughput spectral imaging method that will rival spectral flow cytometry in the analysis of individual cells. We first demonstrated that R-PE subunits have spectroscopic and structural characteristics that make them suitable for SMD. Subunits were isolated from R-PE by high-performance liquid chromatography (HPLC) and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). In addition, R-PE subunits and their enzymatic digests were characterized by several separation and detection methods including HPLC, capillary electrophoresis, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and HPLC-electrospray ionization mass spectrometry (ESI-MS). Favorable absorption and fluorescence of the R-PE subunits and digest peptides originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are covalently attached to cysteine residues. High absorption coefficients and strong fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra in the visible region of electromagnetic spectrum, and relatively low molecular weights make these molecules suitable for use as fluorescence labels of biomolecules and cells. We further designed fluorescent proteins both in vitro and in vivo (in Escherichia coli) based on the highly specific attachment of PEB chromophore to genetically expressed apo-subunits of R-PE. In one example, apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii (P. boldii), and expressed in E. coli. Although expressed apo-subunits formed inclusion

  8. Serum copper and ceruloplasmin levels and urinary copper excretion in thermal injury.

    Science.gov (United States)

    Boosalis, M G; McCall, J T; Solem, L D; Ahrenholz, D H; McClain, C J

    1986-12-01

    Conflicting reports regarding copper status in thermal injury patients have been published. We determined serial serum-copper and serum-ceruloplasmin levels and 24-h urinary excretion of copper in 23 patients with second- and third-degree thermal burns. Throughout hospitalization, mean serum-copper concentration was significantly depressed; lowest levels were found in patients with greater than 40% total body surface area burns. Serum ceruloplasmin was also depressed, an unexpected finding because this protein is a positive acute-phase reactant poststress. Mean urinary excretion of copper was elevated, reaching 2.5 times the upper limit of normal 2 wk postburn. Depressed serum-copper levels paralleled the serum-ceruloplasmin levels rather than the increased urinary-copper losses. Further studies are required to determine the mechanism(s) of this altered copper metabolism and whether physiological or biochemical evidence of copper deficiency accompanies the observed hypocupremia.

  9. Direct Analysis of Reversed-Phase HPTLC Separated Tryptic Protein Digests using a Liquid Microjunction Surface Sampling Probe/ESI-MS System

    Energy Technology Data Exchange (ETDEWEB)

    Emory, Joshua F [ORNL; Walworth, Matthew J [ORNL; Van Berkel, Gary J [ORNL; Schulz, Michael [Merck Research Laboratories; Minarik, susanne [Merck Research Laboratories

    2010-01-01

    The sampling, ionization and detection of tryptic peptides separated in one-dimension on reversed phase HPTLC plates was performed using liquid microjunction surface sampling probe electrospray ionization mass spectrometry. Tryptic digests of five proteins (cytochrome c., myoglobin, beta-casein, lysozyme, and bovine serum albumin) were spotted on reversed phase HPTLC RP-8 F254s and HPTLC RP-18 F254s plates. The plates were then developed using 70/30 methanol/water with 0.1 M ammonium acetate. A dual purpose extraction/electrospray solution containing 70/30/0.1 water/methanol/formic acid was infused through the sampling probe during analysis of the developed lanes. Both full scan mass spectra and data dependent tandem mass spectra were acquired for each development lane to detect and verify the peptide distributions. Data dependent tandem mass spectra provided both protein identification and sequence coverage information. Highest sequence coverages were achieved for cytochrome c. and myoglobin (62.5% and 58.3%, respectively) on reversed phase RP-8 plates. While the tryptic peptides were separated enough for identification, the peptide bands did show some overlap with most peptides located in the lower half of the development lane. Proteins whose peptides were more separated gave higher sequence coverage. Larger proteins such as beta-casein and BSA which were spotted in lower relative amounts gave much lower sequence coverage than the smaller proteins.

  10. New perspectives in protein-based biosensors: the glucokinase from B. stearothermophilus and the odorant-binding protein from C. familiaris as probes for non-consuming analyte sensors

    Science.gov (United States)

    Kuznetsova, Irina M.; Povarova, Olga; Stepanenko, Olesya; Turoverov, Konstantin K.; Crescenzo, Roberta; Varriale, Antonio; Staiano, Maria; D'Auria, Sabato

    2007-06-01

    Glucose sensing and odorant molecules sensing are used as a models to explore the advantages and problems deriving from the use of either enzymes or odorant-binding proteins to develop stable optical biosensors. We report on a novel approach to address the problem of substrate consumption by sensors based on enzymes, namely the utilization of apoenzymes as non-active forms of the protein which are still able to bind the substrate/ligand. We also show studies in which the isolation of an odorant-binding protein from the nose of the dog is used as non-consuming analyte probe for the realization of an integrated optical sensor.

  11. Excimer emission properties on pyrene-labeled protein surface: correlation between emission spectra, ring stacking modes, and flexibilities of pyrene probes.

    Science.gov (United States)

    Fujii, Akira; Sekiguchi, Yutaka; Matsumura, Hiroyoshi; Inoue, Tsuyoshi; Chung, Wen-Sheng; Hirota, Shun; Matsuo, Takashi

    2015-03-18

    The excimer emission of pyrene is popularly employed for investigating the association between pyrene-labeled biomolecules or between pyrene-labeled places in a biomolecule. The property of pyrene excimer emission is affected by the fluctuation in ring stacking modes, which originates from the structural flexibilities of pyrene probes and/or of labeled places. Investigations of the excimer emission in terms of dynamics of pyrene stacking modes provide the detailed spatial information between pyrene-labeled places. In order to evaluate the effects of probe structures and fluctuation in pyrene-pyrene association modes on their emission properties on protein surface, three types of pyrene probe with different linker lengths were synthesized and conjugated to two cysteine residues in the A55C/C77S/V169C mutant of adenylate kinase (Adk), an enzyme that shows a structural transition between OPEN and CLOSED forms. In the CLOSED form of Adk labeled by a pyrene probe with a short linker, excimer emission was found to be predominated by the ground-state association of pyrenes. The pyrene stacking structure on the protein surface was successfully determined by an X-ray crystallographic analysis. However, the emission decay in the protein suggested the existence of several stacking orientations in solution. With the increase in the linker length, the effect of fluctuation in pyrene association modes on the spectral properties distinctly emerged at both ground and excited states. The combination of steady-state and time-resolved spectroscopic analyses is useful for differentiation in the origin of the excimer emission, which is essential for precisely understanding the interaction fashions between pyrene-labeled biomolecules.

  12. In situ activity-based protein profiling of serine hydrolases in E. coli

    Directory of Open Access Journals (Sweden)

    Dmitry Shamshurin

    2014-09-01

    Full Text Available A fluorophosphonate based alkyne activity probe was used for the selective labeling of active serine hydrolases in intact Escherichia coli cells. A biotin-azide tag was subsequently attached to the alkyne functionality of the probe with copper-catalyzed azide-alkyne cycloaddition (CuAAC reaction. Comparison of proteins from in-cell and lysate labeled preparations suggested qualitatively similar patterns of reactivity in both preparations. Approximately 68%, 30 of the total 44 serine hydrolases detectable in E. coli were labeled with the probe indicating significant coverage with a single probe. The methods described here offer a useful tool for profiling and monitoring serine hydrolase activity in situ.

  13. Copper: From neurotransmission to neuroproteostasis

    Directory of Open Access Journals (Sweden)

    Carlos M Opazo

    2014-07-01

    Full Text Available Copper is critical for the Central Nervous System (CNS development and function. In particular, different studies have shown the effect of copper at brain synapses, where it inhibits Long Term Potentation (LTP and receptor pharmacology. Paradoxically, according to recent studies copper is required for a normal LTP response. Copper is released at the synaptic cleft, where it blocks glutamate receptors, which explain its blocking effects on excitatory neurotransmission. Our results indicate that copper also enhances neurotransmission through the accumulation of PSD95 protein, which increase the levels of AMPA receptors located at the plasma membrane of the post-synaptic density. Thus, our findings represent a novel mechanism for the action of copper, which may have implications for the neurophysiology and neuropathology of the CNS. These data indicate that synaptic configuration is sensitive to transient changes in transition metal homeostasis. Our results suggest that copper increases GluA1 subunit levels of the AMPA receptor through the anchorage of AMPA receptors to the plasma membrane as a result of PSD-95 accumulation. Here, we will review the role of copper on neurotransmission of CNS neurons. In addition, we will discuss the potential mechanisms by which copper could modulate neuronal proteostasis (neuroproteostasis in the CNS with focus in the Ubiquitin Proteasome System, which is particularly relevant to neurological disorders such Alzheimer’s disease (AD where copper and protein dyshomeostasis may contribute to neurodegeneration. An understanding of these mechanisms may ultimately lead to the development of novel therapeutic approaches to control metal and synaptic alterations observed in AD patients.

  14. Probing the ability of the coat and vertex protein of the membrane-containing bacteriophage PRD1 to display a meningococcal epitope

    International Nuclear Information System (INIS)

    Huiskonen, Juha T.; Laakkonen, Liisa; Toropainen, Maija; Sarvas, Matti; Bamford, Dennis H.; Bamford, Jaana K.H.

    2003-01-01

    Bacteriophage PRD1 is an icosahedral dsDNA virus with a diameter of 740 A and an outer protein shell composed of 720 copies of major coat protein P3. Spike complexes at the vertices are composed of a pentameric base (protein P31) and a spike structure (proteins P5 and P2) where the N-terminal region of the trimeric P5 is associated with the base and the C-terminal region of P5 is associated with receptor-binding protein P2. The functionality of proteins P3 and P5 was investigated using insertions and deletions. It was observed that P3 did not tolerate changes whereas P5 tolerated changes much more freely. These properties support the hypothesis that viruses have core structures and functions, which remain stable over time, as well as other elements, responsible for host interactions, which are evolutionally more fluid. The insertional probe used was the apex of exposed loop 4 of group B meningococcal outer membrane protein PorA, a medically important subunit vaccine candidate. It was demonstrated that the epitope could be displayed on the virus surface as part of spike protein P5

  15. Use of copper radioisotopes in investigating disorders of copper metabolism

    International Nuclear Information System (INIS)

    Camakaris, J.; Voskoboinik, I.; Brooks, H.; Greenough, M.; Smith, S.; Mercer, J.

    1998-01-01

    Full text: Copper is an essential trace element for life as a number of vital enzymes require it. Copper deficiency can lead to neurological disorders, osteoporosis and weakening of arteries. However Cu is also highly toxic and homeostatic mechanisms have evolved to maintain Cu at levels which satisfy requirements but do not cause toxicity. Toxicity is mediated by the oxidative capacity of Cu and its ability to generate toxic free radicals. There are several acquired and inherited diseases due to either Cu toxicity or Cu deficiency. The study of these diseases facilitates identification of genes and proteins involved in copper homeostasis, and this in turn will provide rational therapeutic approaches. Our studies have focused on Menkes disease in humans which is an inherited and usually lethal copper deficiency. Using copper radioisotopes 64 Cu (t 1/2 = 12.8 hr) and 67 Cu (t 1/2 = 61 hr) we have studied the protein which is mutated in Menkes disease. This is a transmembrane copper pump which is responsible for absorption of copper into the body and also functions to pump out excess Cu from cells when Cu is elevated. It is therefore a vital component of normal Cu homeostasis. We have provided the first biochemical evidence that the Menkes protein functions as a P-type ATPase Cu pump (Voskoboinik et al., FEBS Letters, in press) and these data will be discussed. The assay involved pumping of radiocopper into purified membrane vesicles. Furthermore we have transfected normal and mutant Menkes genes into cells and are carrying out structure-function studies. We are also studying the role of amyloid precursor protein (APP) as a Cu transport protein in order to determine how Cu regulates this protein and its cleavage products. These studies will provide vital information on the relationship between Cu and APP and processes which lead to Alzheimers disease

  16. Development of a generic approach to metalloproteomics; application to the quantitative identification of soluble copper proteins in Escherichia coli

    NARCIS (Netherlands)

    Sevcenco, A.M.; Krijger, G.; Pinkse, M.; Verhaert, P.D.E.M.; Hagen, W.R.; Hagedoorn, P.L.

    2009-01-01

    A combination of techniques to separate and quantify the native proteins associated with a particular transition metal ion from a cellular system has been developed. The procedure involves four steps: (1) labeling of the target proteins with a suitable short-lived radioisotope (suitable isotopes are

  17. Probing the Energetics of Dynactin Filament Assembly and the Binding of Cargo Adaptor Proteins Using Molecular Dynamics Simulation and Electrostatics-Based Structural Modeling.

    Science.gov (United States)

    Zheng, Wenjun

    2017-01-10

    Dynactin, a large multiprotein complex, binds with the cytoplasmic dynein-1 motor and various adaptor proteins to allow recruitment and transportation of cellular cargoes toward the minus end of microtubules. The structure of the dynactin complex is built around an actin-like minifilament with a defined length, which has been visualized in a high-resolution structure of the dynactin filament determined by cryo-electron microscopy (cryo-EM). To understand the energetic basis of dynactin filament assembly, we used molecular dynamics simulation to probe the intersubunit interactions among the actin-like proteins, various capping proteins, and four extended regions of the dynactin shoulder. Our simulations revealed stronger intersubunit interactions at the barbed and pointed ends of the filament and involving the extended regions (compared with the interactions within the filament), which may energetically drive filament termination by the capping proteins and recruitment of the actin-like proteins by the extended regions, two key features of the dynactin filament assembly process. Next, we modeled the unknown binding configuration among dynactin, dynein tails, and a number of coiled-coil adaptor proteins (including several Bicaudal-D and related proteins and three HOOK proteins), and predicted a key set of charged residues involved in their electrostatic interactions. Our modeling is consistent with previous findings of conserved regions, functional sites, and disease mutations in the adaptor proteins and will provide a structural framework for future functional and mutational studies of these adaptor proteins. In sum, this study yielded rich structural and energetic information about dynactin and associated adaptor proteins that cannot be directly obtained from the cryo-EM structures with limited resolutions.

  18. New achievements on biological aspects of copper complexes Casiopeínas®: interaction with DNA and proteins and anti-Trypanosoma cruzi activity.

    Science.gov (United States)

    Becco, Lorena; Rodríguez, Alejandra; Bravo, María Elena; Prieto, María José; Ruiz-Azuara, Lena; Garat, Beatriz; Moreno, Virtudes; Gambino, Dinorah

    2012-04-01

    The mixed-chelate copper(II) complexes Casiopeínas® have been tested in several models in vitro and in vivo, showing promising antitumoral results. However, their mechanism of action remains to be defined. Trying to get a deeper insight into their molecular mode of action, further analyses, including gel electrophoresis, atomic force microscopy and circular dichroism were carried out to study their interaction with DNA and some cytoskeleton proteins. Our results revealed that the interaction of Casiopeínas triggers DNA cleavage by a free radical mechanism. The tested complexes showed a differential response to reducing and scavenger agents. Differences on target preference were also evident using double stranded oligonucleotides as sequence competitors. Surprisingly, distamycin A, a minor groove binder, enhanced the Casiopeínas' action on DNA. On the other hand, the tested Casiopeínas produce strong changes in protein structure of tubulin, integrin and fibronectin. All together these results suggest a multiple mode of action for these metal-based drugs. In addition, since it has been proposed that antitumor drugs efficiently interacting with DNA could also show activity against Trypanosoma cruzi, etiologic agent of Chagas disease, we evaluated the activity of these compounds on this protozoan parasite. The tested complexes showed in vitro anti-T. cruzi activity similar to the anti-trypanosomal reference drug Nifurtimox. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Advanced purification strategy for CueR, a cysteine containing copper(I) and DNA binding protein

    DEFF Research Database (Denmark)

    Balogh, Ria K.; Gyurcsik, Béla; Hunyadi-Gulyás, Éva

    2016-01-01

    . A detailed understanding of their function may be exploited in potential health, environmental and analytical applications. Members of the MerR protein family sense a broad range of mostly late transition and heavy metal ions through their cysteine thiolates. The air sensitivity of latter groups makes......Metal ion regulation is essential for living organisms. In prokaryotes metal ion dependent transcriptional factors, the so-called metalloregulatory proteins play a fundamental role in controlling the concentration of metal ions. These proteins recognize metal ions with an outstanding selectivity......, our procedure consisted of four steps supplemented by DNA digestion. Subsequent anion exchange on Sepharose FF Q 16/10, affinity chromatography on Heparin FF 16/10, second anion exchange on Source 30 Q 16/13 and gel filtration on Superdex 75 26/60 resulted in large amounts of pure CueR protein without...

  20. Evidence that translation reinitiation leads to a partially functional Menkes protein containing two copper-binding sites

    DEFF Research Database (Denmark)

    Paulsen, Marianne; Lund, Connie; Akram, Zarqa

    2006-01-01

    -dependent trafficking of the major part of endogenous and recombinant ATP7A(Delta ex3+ex4) proteins were similar to the wild-type ATP7A protein. Furthermore, the ATP7A(Delta ex3+ex4) cDNA was able to rescue a yeast strain lacking the homologous gene, CCC2. In summary, we propose that reinitiation of the NMD...

  1. Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways.

    Science.gov (United States)

    Anandhan, Annadurai; Rodriguez-Rocha, Humberto; Bohovych, Iryna; Griggs, Amy M; Zavala-Flores, Laura; Reyes-Reyes, Elsa M; Seravalli, Javier; Stanciu, Lia A; Lee, Jaekwon; Rochet, Jean-Christophe; Khalimonchuk, Oleh; Franco, Rodrigo

    2015-09-01

    Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications.

    Science.gov (United States)

    Wei, Hui; Mo, Jingjie; Tao, Li; Russell, Reb J; Tymiak, Adrienne A; Chen, Guodong; Iacob, Roxana E; Engen, John R

    2014-01-01

    The higher order structure of protein therapeutics can be interrogated with hydrogen/deuterium exchange mass spectrometry (HDX-MS). HDX-MS is now a widely used tool in the structural characterization of protein therapeutics. In this review, HDX-MS based workflows designed for protein therapeutic discovery and development processes are presented, focusing on the specific applications of epitope mapping for protein/drug interactions and biopharmaceutical comparability studies. Future trends in the application of HDX-MS in protein therapeutics characterization are also described. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Probing the orientation of surface-immobilized protein G B1 using ToF-SIMS, sum frequency generation, and NEXAFS spectroscopy.

    Science.gov (United States)

    Baugh, Loren; Weidner, Tobias; Baio, J E; Nguyen, Phuong-Cac T; Gamble, Lara J; Stayton, Patrick S; Castner, David G

    2010-11-02

    , with an estimated tilt angle of inner β-strands of 40-50° for both variants (one variant more tilted than the other), consistent with SFG results. The combined results demonstrate the power of using complementary techniques to probe protein orientation on surfaces.

  4. Probing the Orientation of Surface Immobilized Protein G B1 using ToF SIMS, Sum Frequency Generation, and NEXAFS Spectroscopy

    Science.gov (United States)

    Baugh, Loren; Weidner, Tobias; Baio, J.E.; Nguyen, Phuong Cac; Gamble, Lara J.; Stayton, Patrick S.; Castner, David G.

    2010-01-01

    , with an estimated tilt angle of inner β-strands of 40–50° for both variants – one variant more tilted than the other – consistent with SFG results. The combined results demonstrate the power of using complementary techniques to probe protein orientation on surfaces. PMID:20384305

  5. Time-resolved spectroscopy of the probe fluorescence in the study of human blood protein dynamic structure on SR beam

    International Nuclear Information System (INIS)

    Dobretsov, G.E.; Kurek, N.K.; Syrejshchikova, T.I.; Yakimenko, M.N.; Clarke, D.T.; Jones, G.R.; Munro, I.H.

    2000-01-01

    Time-resolved spectroscopy on the SRS of the Daresbury Laboratory was used for the study of the human serum lipoproteins and human blood albumins with fluorescent probes K-37 and K-35, developed in Russia. The probe K-37 was found sensitive to the difference in dynamic properties of the lipid objects. Two sets of the parameters were used for the description of lipid dynamic structure: (1) time-resolved fluorescence spectra and (2) time-resolved fluorescence depolarization as a function of rotational mobility of lipid molecules. Each measured dynamic parameter reflected the monotonous changes of dynamic properties in the range: lipid spheres-very low density lipoproteins-low density lipoproteins-high density lipoproteins-phospholipid liposomes. The range is characterized by the increase of the ratio polar/ nonpolar lipids. Thus, time-resolved fluorescence could be used to detect some structural modifications in lipoproteins related to atherosclerosis and subsequent cardiovascular diseases development

  6. Conjugation reactions in the preparations of quantum dot-based immunoluminescent probes for analysis of proteins by capillary electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Lišková, Marcela; Voráčová, Ivona; Klepárník, Karel; Hezinová, Věra; Přikryl, Jan; Foret, František

    2011-01-01

    Roč. 400, č. 2 (2011), s. 369-379 ISSN 1618-2642 R&D Projects: GA ČR GA203/08/1680; GA MŠk LC06023; GA ČR GAP301/11/2055 Institutional research plan: CEZ:AV0Z40310501 Keywords : CdTe quantum dots * luminescent probes * capillary zone electrophoresis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.778, year: 2011

  7. Target and identify: triazene linker helps identify azidation sites of labelled proteins via click and cleave strategy.

    Science.gov (United States)

    Lohse, Jonas; Schindl, Alexandra; Danda, Natasha; Williams, Chris P; Kramer, Karl; Kuster, Bernhard; Witte, Martin D; Médard, Guillaume

    2017-10-31

    A method for identifying probe modification of proteins via tandem mass spectrometry was developed. Azide bearing molecules are immobilized on functionalised sepharose beads via copper catalysed Huisgen-type click chemistry and selectively released under acidic conditions by chemical cleavage of the triazene linkage. We applied this method to identify the modification site of targeted-diazotransfer on BirA.

  8. Probing the Selectivity and Protein•Protein Interactions of a Non-Reducing Fungal Polyketide Synthase Using Mechanism-Based Crosslinkers

    Science.gov (United States)

    Bruegger, Joel; Haushalter, Bob; Vagstad, Anna; Shakya, Gaurav; Mih, Nathan; Townsend, Craig A.; Burkart, Michael D.; Tsai, Shiou-Chuan

    2013-01-01

    SUMMARY Protein•protein interactions, which often involve interactions between an acyl carrier protein (ACP) and its partner enzymes, are important for coordinating polyketide biosynthesis. However, the nature of such interactions is not well understood, especially in the fungal non-reducing polyketide synthases (NR-PKSs) that biosynthesize toxic and pharmaceutically important polyketides. Here, we employ a mechanism-based crosslinker to successfully probe ACP and ketosynthase (KS) domain interactions in NR-PKSs. We found that crosslinking efficiency is closely correlated with the strength of ACP•KS interactions, and that KS demonstrates strong starter unit selectivity. We further identified positively charged surface residues by KS mutagenesis, which mediate key interactions with the negatively-charged ACP surface. Such complementary/matching contact pairs can serve as “adapter surfaces” for future efforts to generate new polyketides using NR-PKSs. PMID:23993461

  9. COPT6 is a plasma membrane transporter that functions in copper homeostasis in Arabidopsis and is a novel target of SQUAMOSA promoter binding protein-like 7

    Science.gov (United States)

    Among the mechanisms controlling copper homeostasis in plants is the regulation of its uptake and tissue partitioning. Here we characterized a newly identified member of the conserved CTR/COPT family of copper transporters in Arabidopsis thaliana, COPT6. We showed that COPT6 resides at the plasma me...

  10. Evaluation of DNA/Protein interactions and cytotoxic studies of copper(II) complexes incorporated with N, N donor ligands and terpyridine ligand.

    Science.gov (United States)

    Tummalapalli, Kiran; C S, Vasavi; Munusami, Punnagai; Pathak, Madhvesh; M M, Balamurali

    2017-02-01

    A series of four new copper(II) heteroleptic complexes, [Cu(2‴-pytpy) (L)] (NO 3 ) 2 ·2H 2 O (1-4), where 2‴-pytpy=4'-(2'''-Pyridyl)-2, 2':6', 2''-terpyridine, L=bipyridyl (bpy), 1, 10 phenanthroline(phen), dipyridoquinoxaline(dpq) and dipyridophenazine (dppz) were synthesized and characterized by spectroscopic techniques. Further, the molecular structure of the complex (2) was confirmed by single crystal X-ray diffraction technique and the data revealed a penta coordinated, distorted square-pyramidal geometry with triclinic system. The interactions of four complexes with calf thymus DNA and bovine serum albumin (BSA) were investigated by electronic absorption, fluorescence and circular dichroism spectroscopy techniques. Spectral studies substantiated an intercalative binding mode of metal complexes with ct-DNA. Significant binding interactions of the complexes with protein have been further revealed from fluorescence studies. Furthermore, all the four complexes show potential cytotoxicity towards the human liver carcinoma cell line (HepG-2). Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Biological properties of coral GFP-type proteins provide clues for engineering novel optical probes and biosensors

    Science.gov (United States)

    Salih, Anya; Larkum, Anthony W.; Cronin, Thomas W.; Wiedenmann, Joerg; Szymczak, Ron; Cox, Guy C.

    2004-06-01

    In recent years, a variety of Green Fluorescent Protein (GFP)-like pigments have been discovered from corals and other marine organisms. They are widely used to expand the range of available GFP-type proteins in imaging applications, such as in vivo markers for gene expression and protein localization studies, FRET-based (Förster resonance energy transfer) multicolor imaging and biosensors. They have known diverse optical and biochemical properties but their in vivo spectral properties and biological function in marine organisms is only beginning to be understood. We have investigated their spectral diversity, optical properties and cellular microstructure in corals of the Great Barrier Reef with the aim of elucidating their photo-biological function/s as well as to identify novel proteins suitable for GFP-based technologies. We found numerous spectral variants, with emissions covering almost the full range of the visible spectrum. Many of these GFP-like proteins, especially in corals from the more extreme habitats, such as sun-exposed shallows or in deep water, showed a range of light-related spectral characteristics: high photostability, spectral tuning for energy transfer and dynamic photo-induced transformation properties. Intra-cellularly they were organized into spectral donor-acceptor pairs or even arrays, tuned for FRET. Coral color proteins thus offer an exciting potential to expand the use of the available GFPs in bio-imaging applications and as a basis for improved protein engineering.

  12. Cationic spin probe reporting on thermal denaturation and complexation-decomplexation of BSA with SDS. Potential applications in protein purification processes.

    Science.gov (United States)

    Matei, Iulia; Ariciu, Ana Maria; Neacsu, Maria Victoria; Collauto, Alberto; Salifoglou, Athanasios; Ionita, Gabriela

    2014-09-25

    In this work, we present evidence on the suitability of spin probes to report on the thermal treatment of bovine serum albumin (BSA), in the temperature range 293-343 K, and indirectly monitor the release of sodium dodecyl sulfate (SDS) from its complex with BSA using a covalent gel with β-cyclodextrin (β-CD) in the network. The spin probes used, 5- and 7-doxyl-stearic acids (5-DSA, 7-DSA) or 4-(N,N'-dimethyl-N-hexadecyl)ammonium-2,2',6,6'-tetramethylpiperidine-1-oxyl iodide (CAT16), present similar, fatty acid-like structural features. Their continuous wave electron paramagnetic resonance (CW-EPR) spectra, however, reflect different dynamics when complexed with BSA: a restricted motion for 5-DSA, almost nonsensitive to the heating/cooling cycle, and a faster temperature-dependent dynamic motion for CAT16. Molecular docking allows us to rationalize these results by revealing the different binding modes of 5-DSA and CAT16. The EPR data on the temperature effect on BSA are supported by circular dichroism results projecting recovery, upon cooling, of the initial binding ability of BSA for samples heated to 323 K. The interactions occurring in BSA/SDS/β-CD systems are investigated by CW-EPR and FT-ESEEM spectroscopies. It is found that the covalent gel containing β-CD can efficiently remove SDS from the BSA/SDS complex. The gel is not permeable to BSA but it can encapsulate SDS, thus yielding the free protein in solution and allowing recovery of the native protein conformation. Collectively, the accrued knowledge supports potential applications in protein purification biotechnological processes.

  13. Probing the influence of X-rays on aqueous copper solutions using time-resolved in situ combined video/X-ray absorption near-edge/ultraviolet-visible spectroscopy

    NARCIS (Netherlands)

    Mesu, J. Gerbrand; Beale, Andrew M.|info:eu-repo/dai/nl/325802068; de Groot, Frank M. F.|info:eu-repo/dai/nl/08747610X; Weckhuysen, Bert M.|info:eu-repo/dai/nl/285484397

    2006-01-01

    Time-resolved in situ video monitoring and ultraviolet-visible spectroscopy in combination with X-ray absorption near-edge spectroscopy (XANES) have been used for the first time in a combined manner to study the effect of synchrotron radiation on a series of homogeneous aqueous copper solutions in a

  14. An Efficient and Rapid Method to Monitor the Oxidative Degradation of Protein Pharmaceuticals: Probing Tyrosine Oxidation with Fluorogenic Derivatization.

    Science.gov (United States)

    Bommana, Rupesh; Mozziconacci, Olivier; John Wang, Y; Schöneich, Christian

    2017-07-01

    The loss of potency of protein therapeutics can be linked to the oxidation of specific amino acid residues leading to a great variety of oxidative modifications. The comprehensive identification of these oxidative modifications requires high-resolution mass spectrometry analysis, which requires time and expensive resources. Here, we propose a fluorogenic derivatization method of oxidized Tyr and Phe yielding benzoxazole derivatives, as an orthogonal technique for the rapid screening of protein oxidation. Four model proteins, IgG1, human growth hormone (hGH), insulin and bovine serum albumin (BSA) were exposed to oxidation via peroxyl radicals and metal-catalyzed reactions and efficiently screened by fluorogenic derivatization of Tyr and Phe oxidation products. Complementary LC-MS analysis was done to identify the extent of methionine oxidation in oxidized proteins. The Fluorogenic derivatization technique can easily be adapted to a 96-well plate, in which several protein formulations can be screened in short time. Representatively for hGH, we show that the formation of benzoxazole parallels the oxidation of Met to methionine sulfoxide which enables estimation of Met oxidation by just recording the fluorescence. Our rapid fluorescence based screening allows for the fast comparison of the stability of multiple formulations.

  15. Quantum dot bio-conjugate: as a western blot probe for highly sensitive detection of cellular proteins

    Science.gov (United States)

    Kale, Sonia; Kale, Anup; Gholap, Haribhau; Rana, Abhimanyu; Desai, Rama; Banpurkar, Arun; Ogale, Satishchandra; Shastry, Padma

    2012-03-01

    In the present study, we report a quantum dot (QD)-tailored western blot analysis for a sensitive, rapid and flexible detection of the nuclear and cytoplasmic proteins. Highly luminescent CdTe and (CdTe)ZnS QDs are synthesized by aqueous method. High resolution transmission electron microscopy, Raman spectroscopy, fourier transform infrared spectroscopy, fluorescence spectroscopy and X-ray diffraction are used to characterize the properties of the quantum dots. The QDs are functionalized with antibodies of prostate apoptosis response-4 (Par-4), poly(ADP-ribose) polymerases and β actin to specifically bind with the proteins localized in the nucleus and cytoplasm of the cells, respectively. The QD-conjugated antibodies are used to overcome the limitations of conventional western blot technique. The sensitivity and rapidity of protein detection in QD-based approach is very high, with detection limits up to 10 pg of protein. In addition, these labels provide the capability of enhanced identification and localization of marker proteins in intact cells by confocal laser scanning microscopy.

  16. Quantum dot bio-conjugate: as a western blot probe for highly sensitive detection of cellular proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kale, Sonia [Agharkar Research Institute (India); Kale, Anup [University of Alabama, Center for Materials for Information Technology (United States); Gholap, Haribhau; Rana, Abhimanyu [National Chemical Laboratory, Physical and Materials Chemistry Division (India); Desai, Rama [National Centre for Cell Science (India); Banpurkar, Arun [University of Pune, Department of Physics (India); Ogale, Satishchandra, E-mail: sb.ogale@ncl.res.in [National Chemical Laboratory, Physical and Materials Chemistry Division (India); Shastry, Padma, E-mail: padma@nccs.res.in [National Centre for Cell Science (India)

    2012-03-15

    In the present study, we report a quantum dot (QD)-tailored western blot analysis for a sensitive, rapid and flexible detection of the nuclear and cytoplasmic proteins. Highly luminescent CdTe and (CdTe)ZnS QDs are synthesized by aqueous method. High resolution transmission electron microscopy, Raman spectroscopy, fourier transform infrared spectroscopy, fluorescence spectroscopy and X-ray diffraction are used to characterize the properties of the quantum dots. The QDs are functionalized with antibodies of prostate apoptosis response-4 (Par-4), poly(ADP-ribose) polymerases and {beta} actin to specifically bind with the proteins localized in the nucleus and cytoplasm of the cells, respectively. The QD-conjugated antibodies are used to overcome the limitations of conventional western blot technique. The sensitivity and rapidity of protein detection in QD-based approach is very high, with detection limits up to 10 pg of protein. In addition, these labels provide the capability of enhanced identification and localization of marker proteins in intact cells by confocal laser scanning microscopy.

  17. Effect of aqueous media on the copper-ion-mediated phototoxicity of CuO nanoparticles toward green fluorescent protein-expressing Escherichia coli.

    Science.gov (United States)

    Shang, Enxiang; Li, Yang; Niu, Junfeng; Guo, Huiyuan; Zhou, Yijing; Liu, Han; Zhang, Xinqi

    2015-12-01

    Quantitative comparison of different aqueous media on the phototoxicity of copper oxide nanoparticles (CuO NPs) is crucial for understanding their ecological effects. In this study, the phototoxicity of CuO NPs toward the green fluorescent protein-expressing Escherichia coli (GFP-E. coli) under UV irradiation (365 nm) was investigated in Luria-Bertani medium (LB), NaCl solution, deionized water (DI) and phosphate-buffered saline (PBS). The phototoxicity of CuO NPs toward GFP-E. coli decreased in the order of DI>NaCl>PBS>LB because of different released concentrations of Cu(2+). The 3h released Cu(2+) concentrations by 10mg/L CuO NPs in DI water, NaCl solution, LB medium, and PBS were 1946.3 ± 75.6, 1242.5 ± 47.6, 1023.4 ± 41.2, and 1162.1 ± 41.9 μg/L, respectively. Transmission electron microscope and laser scanning confocal microscope images of E. coli exposed to CuO NPs demonstrated that the released Cu(2+) resulted in fragmentation of bacterial cell walls, leakage of intracellular components, and finally death of bacteria in four media after UV light irradiation. In each medium, the bacterial mortality rate logarithmically increased with the releasing concentrations of Cu(2+) by CuO NPs (R(2)>0.90) exposed to 3h UV light. This study highlights the importance of taking into consideration of water chemistry when the phototoxicity of CuO NPs is assessed in nanotoxicity research. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Determination of crude protein in animal feed, forage, grain, and oilseeds by using block digestion with a copper catalyst and steam distillation into boric acid: collaborative study.

    Science.gov (United States)

    Thiex, Nancy J; Manson, Harold; Andersson, Shirley; Persson, Jan-Ake

    2002-01-01

    A collaborative study was conducted to evaluate the repeatability and reproducibility of an extension of AOAC Official Method 991.20, Nitrogen (Crude) in Milk, to animal feed, forage (plant tissue), grain, and oilseed materials. Test portions are digested in an aluminum block at 420 degrees C in sulfuric acid with potassium sulfate and a copper catalyst. Digests are cooled and diluted, and concentrated sodium hydroxide is added to neutralize the acid and make the digest basic; the liberated ammonia is distilled by using steam distillation. The liberated ammonia is trapped in a weak boric acid solution and titrated with a stronger standardized acid, hydrochloric acid; colorimetric endpoint detection is used. Fourteen blind samples were sent to 13 collaborators in the United States, Denmark, Sweden, Germany, and the United Kingdom. Recoveries of nitrogen from lysine, tryptophan, and acetanilide were 86.8, 98.8, and 100.1%, respectively. The within-laboratory relative standard deviation (RSDr, repeatability) ranged from 0.40 to 2.38% for crude protein. The among-laboratories (including within-) relative standard deviation (RSD(R), reproducibility) ranged from 0.44 to 2.38%. It is recommended that the method be adopted First Action by AOAC INTERNATIONAL. A lower concentration (1% H3BO3) of trapping solution was compared with the concentration specified in the original protocol (4% H3BO3) and was found comparable for use in an automatic titration system in which titration begins automatically as soon as distillation starts. The Study Directors recommend that 1% H3BO3 as an optional alternative to 4% boric acid trapping solution be allowed for automatic titrators that titrate throughout the distillation.

  19. The French and North American phenotypes of pyruvate carboxylase deficiency, correlation with biotin containing protein by 3H-biotin incorporation, 35S-streptavidin labeling, and Northern blotting with a cloned cDNA probe.

    OpenAIRE

    Robinson, B H; Oei, J; Saudubray, J M; Marsac, C; Bartlett, K; Quan, F; Gravel, R

    1987-01-01

    Cultured skin fibroblasts from 16 patients with either French or American pyruvate carboxylase (PC) deficiency were examined for their ability to incorporate 3H-biotin into proteins. Cell extracts were also examined for the presence of biotin-containing proteins with 35S-streptavidin, immunoreactive protein with anti-PC antibody, and PC mRNA by Northern blotting with a PC cDNA probe. All the North American presentation patients showed a 3H-biotin protein, a streptavidin protein, and an anti-P...

  20. Probing the role of interfacial waters in protein-DNA recognition using a hybrid implicit/explicit solvation model

    Science.gov (United States)

    Li, Shen; Bradley, Philip

    2013-01-01

    When proteins bind to their DNA target sites, ordered water molecules are often present at the protein-DNA interface bridging protein and DNA through hydrogen bonds. What is the role of these ordered interfacial waters? Are they important determinants of the specificity of DNA sequence recognition, or do they act in binding in a primarily non-specific manner, by improving packing of the interface, shielding unfavorable electrostatic interactions, and solvating unsatisfied polar groups that are inaccessible to bulk solvent? When modeling details of structure and binding preferences, can fully implicit solvent models be fruitfully applied to protein-DNA interfaces, or must the individualistic properties of these interfacial waters be accounted for? To address these questions, we have developed a hybrid implicit/explicit solvation model that specifically accounts for the locations and orientations of small numbers of DNA-bound water molecules while treating the majority of the solvent implicitly. Comparing the performance of this model to its fully implicit counterpart, we find that explicit treatment of interfacial waters results in a modest but significant improvement in protein sidechain placement and DNA sequence recovery. Base-by-base comparison of the performance of the two models highlights DNA sequence positions whose recognition may be dependent on interfacial water. Our study offers large-scale statistical evidence for the role of ordered water for protein DNA recognition, together with detailed examination of several well-characterized systems. In addition, our approach provides a template for modeling explicit water molecules at interfaces that should be extensible to other systems. PMID:23444044

  1. Gold Nanoparticle Aggregation as a Probe of Antifreeze (Glyco) Protein-Inspired Ice Recrystallization Inhibition and Identification of New IRI Active Macromolecules.

    Science.gov (United States)

    Mitchell, Daniel E; Congdon, Thomas; Rodger, Alison; Gibson, Matthew I

    2015-10-26

    Antifreeze (glyco)proteins are found in polar fish species and act to slow the rate of growth of ice crystals; a property known as ice recrystallization inhibition. The ability to slow ice growth is of huge technological importance especially in the cryopreservation of donor cells and tissue, but native antifreeze proteins are often not suitable, nor easily available. Therefore, the search for new materials that mimic this function is important, but currently limited by the low-throughout assays associated with the antifreeze properties. Here 30 nm gold nanoparticles are demonstrated to be useful colorimetric probes for ice recrystallization inhibition, giving a visible optical response and is compatible with 96 well plates for high-throughout studies. This method is faster, requires less infrastructure, and has easier interpretation than the currently used 'splat' methods. Using this method, a series of serum proteins were identified to have weak, but specific ice recrystallization inhibition activity, which was removed upon denaturation. It is hoped that high-throughput tools such as this will accelerate the discovery of new antifreeze mimics.

  2. Probing the effects of cysteine residues on protein adsorption onto gold nanoparticles using wild-type and mutated GB3 proteins.

    Science.gov (United States)

    Siriwardana, Kumudu; Wang, Ailin; Vangala, Karthikeshwar; Fitzkee, Nicholas; Zhang, Dongmao

    2013-09-03

    The role of cysteine residues in the protein binding kinetics and stability on gold nanoparticles (AuNP) was studied using AuNP localized surface plasmon resonance (LSPR) in combination with an organothiol (OT) displacement method. GB3, the third IgG-binding domain of protein G, was used to model protein-AuNP adsorption. While wild-type GB3 (GB30) contains no cysteine residues, bioengineered GB3 variants containing one (GB31) and two (GB32) cysteine residues were also tested. The cysteine content has no significant effect on GB3 binding kinetics with AuNPs, and most protein adsorption occurs within the first few seconds upon protein/AuNP mixing. However, the stability of GB3 on the AuNP surface against OT displacement depends strongly on the cysteine content and the age of the AuNP/GB3 mixture. The GB30 covered AuNPs can be completely destabilized and aggregated by OTs, regardless of the age of the GB30/AuNP mixtures. Long-time incubation of GB31 or GB32 with AuNPs can stabilize AuNPs against the OT adsorption inducted aggregation. This study indicates that multiple forces involved in the GB3/AuNP interaction, and covalent binding between cysteine and AuNP is essential for a stable protein/AuNP complex.

  3. Joining of alumina via copper/niobium/copper interlayers

    Energy Technology Data Exchange (ETDEWEB)

    Marks, Robert A.; Chapman, Daniel R.; Danielson, David T.; Glaeser, Andreas M.

    2000-03-15

    Alumina has been joined at 1150 degrees C and 1400 degrees C using multilayer copper/niobium/copper interlayers. Four-point bend strengths are sensitive to processing temperature, bonding pressure, and furnace environment (ambient oxygen partial pressure). Under optimum conditions, joints with reproducibly high room temperature strengths (approximately equal 240 plus/minus 20 MPa) can be produced; most failures occur within the ceramic. Joints made with sapphire show that during bonding an initially continuous copper film undergoes a morphological instability, resulting in the formation of isolated copper-rich droplets/particles at the sapphire/interlayer interface, and extensive regions of direct bonding between sapphire and niobium. For optimized alumina bonds, bend tests at 800 degrees C-1100 degrees C indicate significant strength is retained; even at the highest test temperature, ceramic failure is observed. Post-bonding anneals at 1000 degrees C in vacuum or in gettered argon were used to assess joint stability and to probe the effect of ambient oxygen partial pressure on joint characteristics. Annealing in vacuum for up to 200 h causes no significant decrease in room temperature bend strength or change in fracture path. With increasing anneal time in a lower oxygen partial pressure environment, the fracture strength decreases only slightly, but the fracture path shifts from the ceramic to the interface.

  4. Probing the molecular forces involved in binding of selected volatile flavour compounds to salt-extracted pea proteins.

    Science.gov (United States)

    Wang, Kun; Arntfield, Susan D

    2016-11-15

    Molecular interactions between heterologous classes of flavour compounds with salt-extracted pea protein isolates (PPIs) were determined using various bond disrupting agents followed by GC/MS analysis. Flavour bound by proteins decreased in the order: dibutyl disulfide>octanal>hexyl acetate>2-octanone=benzaldehyde. Benzaldehyde, 2-octanone and hexyl acetate interacted non-covalently with PPIs, whereas octanal bound PPIs via covalent and non-covalent forces. Dibutyl disulfide reacted with PPIs covalently, as its retention was not diminished by urea and guanidine hydrochloride. Using propylene glycol, H-bonding and ionic interactions were implicated for hexyl acetate, benzaldehyde, and 2-octanone. A protein-destabilising salt (Cl3CCOONa) reduced bindings for 2-octanone, hexyl acetate, and benzaldehyde; however, retention for octanal and dibutyl disulfide increased. Conversely, a protein-stabilising salt (Na2SO4) enhanced retention for benzaldehyde, 2-octanone, hexyl acetate and octanal. Formation of a volatile flavour by-product, 1-butanethiol, from dibutyl disulfide when PPIs were treated with dithiothreitol indicated occurrence of sulfhydryl-disulfide interchange reactions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Does an infrasonic acoustic shock wave resonance of the manganese 3+ loaded/copper depleted prion protein initiate the pathogenesis of TSE?

    Science.gov (United States)

    Purdey, Mark

    2003-06-01

    Intensive exposures to natural and artificial sources of infrasonic acoustic shock (tectonic disturbances, supersonic aeroplanes, etc.) have been observed in ecosystems supporting mammalian populations that are blighted by clusters of traditional and new variant strains of transmissible spongiform encephalopathy (TSE). But TSEs will only emerge in those 'infrasound-rich' environments which are simultaneously influenced by eco-factors that induce a high manganese (Mn)/low copper (Cu)-zinc (Zn) ratio in brains of local mammalian populations. Since cellular prion protein (PrPc) is a cupro-protein expressed throughout the circadian mediated pathways of the body, it is proposed that PrP's Cu component performs a role in the conduction and distribution of endogenous electromagnetic energy; energy that has been transduced from incoming ultraviolet, acoustic, geomagnetic radiations. TSE pathogenesis is initiated once Mn substitutes at the vacant Cu domain on PrPc and forms a nonpathogenic, protease resistant, 'sleeping' prion. A second stage of pathogenesis comes into play once a low frequency wave of infrasonic shock metamorphoses the piezoelectric atomic structure of the Mn 3+ component of the prion, thereby 'priming' the sleeping prion into its fully fledged, pathogenic TSE isoform - where the paramagnetic status of the Mn 3+ atom is transformed into a stable ferrimagnetic lattice work, due to the strong electron-phonon coupling resulting from the dynamic 'Jahn-Teller' type distortions of the oxygen octahedra specific to the trivalent Mn species. The so called 'infectivity' of the prion is a misnomer and should be correctly defined as the contagious field inducing capacity of the ferrimagnetic Mn 3+ component of the prion; which remains pathogenic at all temperatures below the 'curie point'. A progressive domino-like 'metal to ligand to metal' ferrimagnetic corruption of the conduits of electromagnetic superexchange is initiated. The TSE diseased brain can be likened to

  6. Copper Resistance of the Emerging Pathogen Acinetobacter baumannii.

    Science.gov (United States)

    Williams, Caitlin L; Neu, Heather M; Gilbreath, Jeremy J; Michel, Sarah L J; Zurawski, Daniel V; Merrell, D Scott

    2016-10-15

    Acinetobacter baumannii is an important emerging pathogen that is capable of causing many types of severe infection, especially in immunocompromised hosts. Since A. baumannii can rapidly acquire antibiotic resistance genes, many infections are on the verge of being untreatable, and novel therapies are desperately needed. To investigate the potential utility of copper-based antibacterial strategies against Acinetobacter infections, we characterized copper resistance in a panel of recent clinical A. baumannii isolates. Exposure to increasing concentrations of copper in liquid culture and on solid surfaces resulted in dose-dependent and strain-dependent effects; levels of copper resistance varied broadly across isolates, possibly resulting from identified genotypic variation among strains. Examination of the growth-phase-dependent effect of copper on A. baumannii revealed that resistance to copper increased dramatically in stationary phase. Moreover, A. baumannii biofilms were more resistant to copper than planktonic cells but were still susceptible to copper toxicity. Exposure of bacteria to subinhibitory concentrations of copper allowed them to better adapt to and grow in high concentrations of copper; this copper tolerance response is likely achieved via increased expression of copper resistance mechanisms. Indeed, genomic analysis revealed numerous putative copper resistance proteins that share amino acid homology to known proteins in Escherichia coli and Pseudomonas aeruginosa Transcriptional analysis revealed significant upregulation of these putative copper resistance genes following brief copper exposure. Future characterization of copper resistance mechanisms may aid in the search for novel antibiotics against Acinetobacter and other highly antibiotic-resistant pathogens. Acinetobacter baumannii causes many types of severe nosocomial infections; unfortunately, some isolates have acquired resistance to almost every available antibiotic, and treatment options

  7. Pt(CN)42- and Au(CN)2-: potential general probes for anion-binding sites of proteins

    International Nuclear Information System (INIS)

    Norne, J.E.; Lilja, H.; Lindman, B.; Einarsson, R.; Zeppezauer, M.

    1975-01-01

    Nuclear magnetic quadrupole relaxation appears to be a general method for studying the binding of anions to proteins. This is shown by the increase in transverse quadrupole relaxation rate of 35 Cl - and 81 Br - in the presence of horse liver alcohol dehydrogenase, lysozyme, trypsin, α-chymotrypsin, human carbonic anhydrase, fructose-1.6-biphosphate aldolase and human serum albumin. Of the many possible binding sites at the surface of a protein (e.g. positively charged amino acid side-chains) only a few account for the main part of the relaxation enhancement. This is shown by the decrease in 35 Cl - and 81 Br - relaxation rate on addition of functional ligands. (orig./HK) [de

  8. Population size dependence of fitness effect distribution and substitution rate probed by biophysical model of protein thermostability

    OpenAIRE

    Goldstein, R. A.

    2013-01-01

    The predicted effect of effective population size on the distribution of fitness effects and substitution rate is critically dependent on the relationship between sequence and fitness. This highlights the importance of using models that are informed by the molecular biology, biochemistry, and biophysics of the evolving systems. We describe a computational model based on fundamental aspects of biophysics, the requirement for (most) proteins to be thermodynamically stable. Using this model, we ...

  9. Cultural probes

    DEFF Research Database (Denmark)

    Madsen, Jacob Østergaard

    The aim of this study was thus to explore cultural probes (Gaver, Boucher et al. 2004), as a possible methodical approach, supporting knowledge production on situated and contextual aspects of occupation.......The aim of this study was thus to explore cultural probes (Gaver, Boucher et al. 2004), as a possible methodical approach, supporting knowledge production on situated and contextual aspects of occupation....

  10. Characterization of Caco-2 cells stably expressing the protein-based zinc probe eCalwy-5 as a model system for investigating intestinal zinc transport.

    Science.gov (United States)

    Maares, Maria; Keil, Claudia; Thomsen, Susanne; Günzel, Dorothee; Wiesner, Burkhard; Haase, Hajo

    2018-01-29

    Intestinal zinc resorption, in particular its regulation and mechanisms, are not yet fully understood. Suitable intestinal cell models are needed to investigate zinc uptake kinetics and the role of labile zinc in enterocytes in vitro. Therefore, a Caco-2 cell clone was produced, stably expressing the genetically encoded zinc biosensor eCalwy-5. The aim of the present study was to reassure the presence of characteristic enterocyte-specific properties in the Caco-2-eCalwy clone. Comparison of Caco-2-WT and Caco-2-eCalwy cells revealed only slight differences regarding subcellular localization of the tight junction protein occludin and alkaline phosphatase activity, which did not affect basic integrity of the intestinal barrier or the characteristic brush border membrane morphology. Furthermore, introduction of the additional zinc-binding protein in Caco-2 cells did not alter mRNA expression of the major intestinal zinc transporters (zip4, zip5, znt-1 and znt-5), but increased metallothionein 1a-expression and cellular resistance to higher zinc concentrations. Moreover, this study examines the effect of sensor expression level on its saturation with zinc. Fluorescence cell imaging indicated considerable intercellular heterogeneity in biosensor-expression. However, FRET-measurements confirmed that these differences in expression levels have no effect on fractional zinc-saturation of the probe. Copyright © 2018 Elsevier GmbH. All rights reserved.

  11. In-capillary probing of quantum dots and fluorescent protein self-assembly and displacement using Förster resonance energy transfer.

    Science.gov (United States)

    Wang, Jianhao; Fan, Jie; Li, Jinchen; Liu, Li; Wang, Jianpeng; Jiang, Pengju; Liu, Xiaoqian; Qiu, Lin

    2017-02-01

    Herein, a Förster resonance energy transfer system was designed, which consisted of CdSe/ZnS quantum dots donor and mCherry fluorescent protein acceptor. The quantum dots and the mCherry proteins were conjugated to permit Förster resonance energy transfer. Capillary electrophoresis with fluorescence detection was used for the analyses for the described system. The quantum dots and mCherry were sequentially injected into the capillary, while the real-time fluorescence signal of donor and acceptor was simultaneously monitored by two channels with fixed wavelength detectors. An effective separation of complexes from free donor and acceptor was achieved. Results showed quantum dots and hexahistidine tagged mCherry had high affinity and the assembly was affected by His 6 -mCherry/quantum dot molar ratio. The kinetics of the self-assembly was calculated using the Hill equation. The microscopic dissociation constant values for out of- and in-capillary assays were 10.49 and 23.39 μM, respectively. The capillary electrophoresis with fluorescence detection that monitored ligands competition assay further delineated the different binding capacities of histidine containing peptide ligands for binding sites on quantum dots. This work demonstrated a novel approach for the improvement of Förster resonance energy transfer for higher efficiency, increased sensitivity, intuitionistic observation, and low sample requirements of the in-capillary probing system. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Population size dependence of fitness effect distribution and substitution rate probed by biophysical model of protein thermostability.

    Science.gov (United States)

    Goldstein, Richard A

    2013-01-01

    The predicted effect of effective population size on the distribution of fitness effects and substitution rate is critically dependent on the relationship between sequence and fitness. This highlights the importance of using models that are informed by the molecular biology, biochemistry, and biophysics of the evolving systems. We describe a computational model based on fundamental aspects of biophysics, the requirement for (most) proteins to be thermodynamically stable. Using this model, we find that differences in population size have minimal impact on the distribution of population-scaled fitness effects, as well as on the rate of molecular evolution. This is because larger populations result in selection for more stable proteins that are less affected by mutations. This reduction in the magnitude of the fitness effects almost exactly cancels the greater selective pressure resulting from the larger population size. Conversely, changes in the population size in either direction cause transient increases in the substitution rate. As differences in population size often correspond to changes in population size, this makes comparisons of substitution rates in different lineages difficult to interpret.

  13. Separation of copper-64 from copper phthalocyanine

    International Nuclear Information System (INIS)

    Battaglin, R.I.M.

    1979-01-01

    The separation of copper-64 from irradiated copper phthalocyanine by Szilard-Chalmers effect is studied. Two methods of separation are used: one of them is based on the dissolution of the irradiated dry compound in concentrated sulfuric acid following its precipitation in water. In the other one the compound is irradiated with water in paste form following treatment with water and hydrochloric acid. The influence of the crystal form of the copper phthalocyanine on the separation yield of copper-64 is shown. Preliminary tests using the ionic exchange technique for purification and changing of copper-64 sulfate to chloride form are carried out. The specific activity using the spectrophotometric technique, after the determination of the copper concentration in solution of copper-64, is calculated. (Author) [pt

  14. A simplistic approach to green future with eco-friendly luminescent carbon dots and their application to fluorescent nano-sensor 'turn-off' probe for selective sensing of copper ions.

    Science.gov (United States)

    Das, Poushali; Ganguly, Sayan; Bose, Madhuparna; Mondal, Subhadip; Das, Amit Kumar; Banerjee, Susanta; Das, Narayan Chandra

    2017-06-01

    Zero-dimensional fluorescent nanoparticles having specificity as molecular probe appears to be strategically balanced fluorescent nano-probes. In this work, purified lemon extract and l-arginine have been thermally coupled for the extremely acute detection of Cu 2+ in aqueous medium. The Cu 2+ ions may be captured by the amino groups on the surface of the nano-sensor to form cupric ammine complex resulting in quenched fluorescence via an inner filter effect. Our proposed nano-probe is N-doped carbon dots (NCDs) which are efficiently selective as fluorescent chemosensor due to enormous binding affinity towards Cu 2+ in a wide range of concentration (0.05-300μM) within a few minutes. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Mononuclear copper(II) complexes with 3,5-substituted-4-salicylidene-amino-3,5-dimethyl-1,2,4-triazole: synthesis, structure and potent inhibition of protein tyrosine phosphatases.

    Science.gov (United States)

    Ma, Ling; Lu, Liping; Zhu, Miaoli; Wang, Qingming; Li, Ying; Xing, Shu; Fu, Xueqi; Gao, Zengqiang; Dong, Yuhui

    2011-06-28

    Six copper complexes of Schiff base ligands containing 3,5-substituted-4-salicylideneamino-3,5-dimethyl-1,2,4-triazole have been synthesized and well characterized. The structures of complexes 1 and 2 were determined by X-ray crystal analysis. Fluorescence and potentiometric study indicated that in the physiological pH range, one ligand was dissociated from the complexes to form 1:1 mononucleus copper complexes. The complexes potently inhibit protein tyrosine phosphatase 1B (PTP1B), T-cell protein tyrosine phosphatase (TCPTP), megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2) and Src homology phosphatase 1 (SHP-1) with 3-4 fold selectivity against PTP1B over TCPTP and PTP-MEG2, and 3-9 fold over SHP-1, but display almost no inhibition against Src homology phosphatase 2 (SHP-2). Complex 1 inhibits PTP1B with a competitive model with K(i) of 30 nM. Substitution with small groups at the phenyl of the ligand does not obviously influence the inhibitory ability of the complexes. This journal is © The Royal Society of Chemistry 2011

  16. Copper in plants

    OpenAIRE

    Yruela, Inmaculada

    2005-01-01

    Copper is an essential metal for normal plant growth and development, although it is also potentially toxic. Copper participates in numerous physiological processes and is an essential cofactor for many metalloproteins, however, problems arise when excess copper is present in cells. Excess copper inhibits plant growth and impairs important cellular processes (i.e., photosynthetic electron transport). Since copper is both an essential cofactor and a toxic element, involving a complex network o...

  17. Bioreduction of Cu(II) by cell-free copper reductase from a copper resistant Pseudomonas sp. NA.

    Science.gov (United States)

    Andreazza, Robson; Okeke, Benedict C; Pieniz, Simone; Brandelli, Adriano; Lambais, Mácio R; Camargo, Flávio A O

    2011-11-01

    Environmental copper contamination is a serious human health problem. Copper reductase is produced by microorganisms to facilitate copper uptake by ATPases into the cells increasing copper biosorption. This study assessed the reduction of Cu(II) by cell-free extracts of a highly copper-resistant bacterium, Pseudomonas sp. strain NA, isolated from vineyard soil contaminated with copper. Both intact cells and cell-free extract of Pseudomonas sp. strain NA displayed substantial reduction of Cu(II). Intact cells reduced more then 80 mg L(-1) of Cu(II) from medium amended with 200 mg L(-1) of copper after 24 h of incubation. Cell-free extract of the isolate reduced more than 65% of the Cu(II) at initial copper concentration of 200 mg L(-1) after 24 h. Soluble protein production was high at 72 h of incubation at 100 mg L(-1) of copper, with more then 60 μg L(-1) of total soluble protein in cell-free extract recorded. Cu(II) reduction by isolate NA was increased when copper concentration increased for both intact cells and cell-free extract. Results indicate that Pseudomonas sp. strain NA produces copper reductase enzyme as the key mechanism of copper biotransformation.

  18. Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2

    DEFF Research Database (Denmark)

    Litman, Thomas; Jensen, Ulla; Hansen, Alastair

    2002-01-01

    Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides...... corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed...... membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP...

  19. Photoactivation of the BLUF protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues

    KAUST Repository

    Gil, Agnieszka A.

    2017-09-06

    The flavin chromophore in blue light using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppA by the introduction of fluorotyrosine (F-Tyr) analogs that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark to light adapted form) photoreaction was observed, the change in Y21 pKa led to a 4,000-fold increase in the rate of dark state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppA, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppA, despite their sharing highly conserved FAD binding architectures.

  20. Mobile probes

    DEFF Research Database (Denmark)

    Ørngreen, Rikke; Jørgensen, Anna Neustrup; Noesgaard, Signe Schack

    2016-01-01

    to in an interview. This method provided valuable insight into the contextual use, i.e. how did the online resource transfer to the work practice. However, the research team also found that mobile probes may provide the scaffolding necessary for individual and peer learning at a very local (intra-school) community...... level. This paper is an initial investigation of how the mobile probes process proved to engage teachers in their efforts to improve teaching. It also highlights some of the barriers emerging when applying mobile probes as a scaffold for learning.......A project investigating the effectiveness of a collection of online resources for teachers' professional development used mobile probes as a data collection method. Teachers received questions and tasks on their mobile in a dialogic manner while in their everyday context as opposed...

  1. Imidazole C-2 Hydrogen/Deuterium Exchange Reaction at Histidine for Probing Protein Structure and Function with MALDI Mass Spectrometry

    Science.gov (United States)

    Hayashi, Naoka; Kuyama, Hiroki; Nakajima, Chihiro; Kawahara, Kazuki; Miyagi, Masaru; Nishimura, Osamu; Matsuo, Hisayuki; Nakazawa, Takashi

    2015-01-01

    We present a mass spectrometric method for analyzing protein structure and function, based on the imidazole C-2 or histidine Cε1 hydrogen/deuterium (H/D) exchange reaction, which is intrinsically second order with respect to the concentrations of the imidazolium cation and OD− in D2O. The second-order rate constant (k2) of this reaction was calculated from the pH-dependency of the pseudo-first-order rate constant (kφ) obtained from the change of average mass ΔMr (0 ≤ ΔMr exchange rate in terms of log(k2max/k2) representing the deviation of k2 from k2max. In the catalytic site of bovine ribonuclease A, His12 showed much larger change in log(k2max/k2) compared with His119 upon binding with cytidine 3′-monophosphate, as anticipated from the X-ray structures and the possible change in solvent accessibility. However, there is a need of considering the hydrogen bonds of the imidazole group with non-dissociable groups to interpret an extremely slow H/D exchange rate of His48 in partially solvent-exposed situation. PMID:24606199

  2. Counting probe

    International Nuclear Information System (INIS)

    Matsumoto, Haruya; Kaya, Nobuyuki; Yuasa, Kazuhiro; Hayashi, Tomoaki

    1976-01-01

    Electron counting method has been devised and experimented for the purpose of measuring electron temperature and density, the most fundamental quantities to represent plasma conditions. Electron counting is a method to count the electrons in plasma directly by equipping a probe with the secondary electron multiplier. It has three advantages of adjustable sensitivity, high sensitivity of the secondary electron multiplier, and directional property. Sensitivity adjustment is performed by changing the size of collecting hole (pin hole) on the incident front of the multiplier. The probe is usable as a direct reading thermometer of electron temperature because it requires to collect very small amount of electrons, thus it doesn't disturb the surrounding plasma, and the narrow sweep width of the probe voltage is enough. Therefore it can measure anisotropy more sensitively than a Langmuir probe, and it can be used for very low density plasma. Though many problems remain on anisotropy, computer simulation has been carried out. Also it is planned to provide a Helmholtz coil in the vacuum chamber to eliminate the effect of earth magnetic field. In practical experiments, the measurement with a Langmuir probe and an emission probe mounted to the movable structure, the comparison with the results obtained in reverse magnetic field by using a Helmholtz coil, and the measurement of ionic sound wave are scheduled. (Wakatsuki, Y.)

  3. Mycobacterium tuberculosis and Copper: A Newly Appreciated Defense against an Old Foe?*

    Science.gov (United States)

    Darwin, K. Heran

    2015-01-01

    Several independent studies have recently converged upon the conclusion that the human bacterial pathogen Mycobacterium tuberculosis encounters copper during infections. At least three independently regulated pathways respond to excess copper and are required for the full virulence of M. tuberculosis in animals. In this review, I will discuss the functions of the best-characterized copper-responsive proteins in M. tuberculosis, the potential sources of copper during an infection, and remaining questions about the interface between copper and tuberculosis. PMID:26055711

  4. /sup 31/P NMR probes of sipunculan erythrocytes containing the O/sub 2/-carying protein hemerythrin

    Energy Technology Data Exchange (ETDEWEB)

    Robitaille, P.M.L.; Kurtz, D.M. Jr

    1988-06-14

    Reported are the first examinations by /sup 31/P NMR of erythrocytes containing the non-heme iron O/sub 2/ carrying protein hemerythrin (Hr). Intact coelomic erythrocytes from the sipunculids Phascolopsis gouldii and Themiste zostericola were shown by /sup 31/P NMR to contain O-phosphorylethanolamine and 2-amino-ethylphosphonate as the major soluble phosphorus metabolites. This combination of major metabolites appears to be unique to sipunculan erythrocytes. Nucleoside triphosphates and mannose 1-phosphate were present in lower concentrations. The concentration of O-phosphorylethanolamine within P. gouldii erythrocytes was established to be > 20 mM. T. zostericola erythrocytes contained relatively high levels of 2-amino-ethylphosphonate and lower levels of O-phosphorylethanolamine compared with those of P. gouldii. For P. gouldii and T. zostericola the intracellular pHs were determined to be 7.2 +/- 0.1 and 7.1 +/- 0.1, respectively, in air-equilibrated erythrocytes, and 6.5 +/- 0.1 in anaerobic P. gouldii erythrocytes. O-Phosphorylethanolamine was found to bind weakly to P. gouldii metHr. This interaction is best characterized by either negative cooperativity or nonspecific binding. O-phosphorylethanolamine strongly inhibits azide binding to the iron site of P. gouldii metHr at pH 7.2. The rate of azide binding decreases by approx. 85-fold in the presence of 0.33 M O-phosphorylethanolamine. However, neither O-phosphorylethanolamine nor 2-aminoethylphosphonate at 0.33 M was found to have any significant effect on O/sub 2/ affinity of P. gouldii deoxyHr. Alternative functions for the two metabolites are suggested.

  5. [The changes of processes of free radical oxidation of lipids and proteins, antioxidant defence in rats with hypofunction of the thyroid gland in conditions of iodine and copper deficiency].

    Science.gov (United States)

    Voronych-Semchenko, N M; Huranych, T V

    2014-01-01

    Thyroid status, copper balance, correlation of processes of peroxide oxidation of lipids (POL), proteins (POP), antioxidant defence (AOD) were examined in experiments on rats with hypofunction of thyroid gland under iodine monodeficit (HTGI) and combined iodine and copper deficit (HTGI+Cu). It was determined that a combined deficit of microelements is accompanied by a distribution of copper content between different tissues (increase in red blood cell mass and cerebrum, decrease in myocardium), essential changes of indexes of hypotalamo-hypophysis-thyroid axis, oxygen-dependent metabolism, antiradical defense, exacerbating the effects of negative influence of each of them on organism. It was established that HTGI+Cu causes a suppression of oxygen-dependent processes. In thyroid gland, it is shown a decrease of content of dyenic conjugates (DC) by 69,70% , of TBA-reacting products (TBA-RP) by 47,72% in diencephalon, the volume of modified proteins (VMP) - by 37,10-98,98% in the tissues of diencephalons. The results obtained let us to suggest a pivotal role ofmicroelement dysbalance and metabolic mechanisms in pathogenesis of cardiological pathology under thyroid dysfunction. The development of HTGI +Cu exhausts the resources of AOD: decreases the activity of catalase (on 47,05%), superoxide dismutase (on 33,13%), ceruloplasmine (on 33,93%) and saturation of transferrin with iron (on 56,76%) against the background of selective rise in the activity of glu-tationreductase (in 2,8 time) in comparison with the control data. The long-term disturbances ofantyoxidative defence can be the reason of manifestation of oxygendependent processes and the development of pathological changes in separate physiological systems of organism.

  6. Increased type I collagen content and DNA binding activity of a single-stranded, cytosine-rich sequence in the high-salt buffer protein extract of the copper-deficient rat heart.

    Science.gov (United States)

    Zeng, Huawei; Saari, Jack T

    2004-11-01

    Dietary copper (Cu) deficiency not only causes a hypertrophic cardiomyopathy but also increases cancer risk in rodent models. However, a possible alteration in gene expression has not been fully examined. The present study was undertaken to determine the effect of Cu deficiency on protein profiles in rat heart tissue. Male Sprague-Dawley rats were fed diets that were either a Cu-adequate diet (6.0 microg Cu/g diet, n = 6) or a Cu-deficient diet (0.3 microg Cu/g diet, n = 6) for 5 weeks. The high-salt buffer (HSB) protein extract from heart tissue of Cu-deficient, but not Cu-adequate rats showed a 132 kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. This protein band stained pink with Coomassie Blue, suggesting the presence of collagens or other proline-rich proteins. Dot immunoblotting demonstrated that total type I collagen was increased by 110% in HSB protein extract from Cu-deficient, relative to Cu-adequate, rats. Liquid chromatography with mass spectrometry analysis indicated that the 132 kDa protein band contained a collagen alpha (I) chain precursor as well as a leucine-rich protein 130 (LRP130) in HSB protein extract from Cu-deficient but not Cu-adequate rats. A gel shift assay showed that HSB protein extract from Cu-deficient rats bound to a single-stranded cytosine-rich DNA with higher affinity than the extract of Cu-adequate rats, similar to reports of an increase in LRP130 single-stranded DNA binding activity in several types of tumor cells. Collectively, these results not only suggest an additional feature of altered collagen metabolism with Cu deficiency but also demonstrate for the first time an increase in single-stranded cytosine-rich DNA binding in Cu-deficient rat heart.

  7. Synthesis and characterization of tricarbonyl-Re/Tc(I chelate probes targeting the G protein-coupled estrogen receptor GPER/GPR30.

    Directory of Open Access Journals (Sweden)

    Ritwik Burai

    Full Text Available The discovery of the G protein-coupled estrogen receptor GPER (also GPR30 and the resulting development of selective chemical probes have revealed new aspects of estrogen receptor biology. The potential clinical relevance of this receptor has been suggested from numerous studies that have identified GPER expression in breast, endometrial, ovarian and other cancers. Thus GPER can be considered a candidate biomarker and target for non-invasive imaging and therapy. We have designed and synthesized a series of organometallic tricarbonyl-rhenium complexes conjugated to a GPER-selective small molecule derived from tetrahydro-3H-cyclopenta[c]quinoline. The activity and selectivity of these chelates in GPER-mediated signaling pathways were evaluated. These results demonstrate that GPER targeting characteristics depend strongly on the structure of the chelate and linkage. Ethanone conjugates functioned as agonists, a 1,2,3-triazole spacer yielded an antagonist, and derivatives with increased steric volume exhibited decreased activities. Promising GPER selectivity was observed, as none of the complexes interacted with the nuclear estrogen receptors. Radiolabeling with technetium-99m in aqueous media was efficient and gave radioligands with high radiochemical yields and purity. These chelates have favorable physicochemical properties, show excellent stability in biologically relevant media, exhibit receptor specificity and are promising candidates for continuing development as diagnostic imaging agents targeting GPER expression in cancer.

  8. Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer

    Science.gov (United States)

    Godet, Julien; Kenfack, Cyril; Przybilla, Frédéric; Richert, Ludovic; Duportail, Guy; Mély, Yves

    2013-01-01

    The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (−)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11–55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger–dependent binding of NC to the G10 and G50 residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G10 and G50 the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway. PMID:23511968

  9. Probing and improving student's understanding of protein α-helix structure using targeted assessment and classroom interventions in collaboration with a faculty community of practice.

    Science.gov (United States)

    Loertscher, Jennifer; Villafañe, Sachel M; Lewis, Jennifer E; Minderhout, Vicky

    2014-01-01

    The increasing availability of concept inventories and other assessment tools in the molecular life sciences provides instructors with myriad avenues to probe student understanding. For example, although molecular visualization is central to the study of biochemistry, a growing body of evidence suggests that students have substantial limitations in their ability to recognize and interpret basic features of biological macromolecules. In this study, a pre/posttest administered to students at diverse institutions nationwide revealed a robust incorrect idea about the location of the amino acid side chains in the protein α-helix structure. Because this incorrect idea was present even after a semester of biochemistry instruction at a range of institutions, an intervention was necessary. A community of expert biochemistry instructors collaborated to design two active learning classroom activities that systematically examine α-helix structure and function. Several participating faculty used one or both of the activities in their classrooms and some improvement of student understanding of this concept was observed. This study provides a model of how a community of instructors can work together using assessment data to inform targeted changes in instruction with the goal of improving student understanding of fundamental concepts. Copyright © 2014 by The International Union of Biochemistry and Molecular Biology.

  10. [Effect of copper deficiency on iron metabolism in rats].

    Science.gov (United States)

    Wang, Kebo; Wang, Chaoxu; Liu, Baosheng; Jiang, Shan

    2010-07-01

    To study the effect of copper deficiency on the nutritional status of iron, the expression of hepcidin mRNA and transferrin receptor mRNA in rats. Forty eight clean male SD rats were randomly divided into four groups according to body weight; and there were 12 rats in each group. The groups are normal iron and copper control group (group I), normal iron and copper deficiency group (group II), normal iron and copper slightly deficient group (group III), both iron and copper slightly deficient group (group IV). Serum, liver and spleen of rats were collected by the end of 8th week. Serum copper, serum iron, hemoglobin, serum transferrin receptor, serum ferritin, liver iron and liver copper, spleen iron and spleen copper were determined. The expression of liver transferring receptor mRNA and hepcidin mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR) method. Compared with the controls, the contents of serum iron and serum ferritin decreased (P copper deficiency. The expression of transferrin receptor mRNA in liver increased but the expression of hepcidin mRNA in liver decreased significantly under copper deficiency (P copper deficiency through influencing the absorption, storage and transportation of iron. Under the condition of copper deficiency, the expression of hepcidin mRNA in liver was lowered and the expression of transferrin receptor mRNA was enhanced through the way of iron response element-iron regulatory protein (IRE-IRP) to regulate iron metabolism.

  11. DNA probes

    International Nuclear Information System (INIS)

    Castelino, J.

    1992-01-01

    The creation of DNA probes for detection of specific nucleotide segments differs from ligand detection in that it is a chemical rather than an immunological reaction. Complementary DNA or RNA is used in place of the antibody and is labelled with 32 P. So far, DNA probes have been successfully employed in the diagnosis of inherited disorders, infectious diseases, and for identification of human oncogenes. The latest approach to the diagnosis of communicable and parasitic infections is based on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is encoded by DNA and DNA probe approach to identification of pathogens is unique because the focus of the method is the nucleic acid content of the organism rather than the products that the nucleic acid encodes. Since every properly classified species has some unique nucleotide sequences that distinguish it from every other species, each organism's genetic composition is in essence a finger print that can be used for its identification. In addition to this specificity, DNA probes offer other advantages in that pathogens may be identified directly in clinical specimens

  12. The Zygosaccharomyces bailii transcription factor Haa1 is required for acetic acid and copper stress responses suggesting subfunctionalization of the ancestral bifunctional protein Haa1/Cup2.

    Science.gov (United States)

    Palma, Margarida; Dias, Paulo Jorge; Roque, Filipa de Canaveira; Luzia, Laura; Guerreiro, Joana Fernandes; Sá-Correia, Isabel

    2017-01-13

    The food spoilage yeast species Zygosaccharomyces bailii exhibits an extraordinary capacity to tolerate weak acids, in particular acetic acid. In Saccharomyces cerevisiae, the transcription factor Haa1 (ScHaa1) is considered the main player in genomic expression reprogramming in response to acetic acid stress, but the role of its homologue in Z. bailii (ZbHaa1) is unknown. In this study it is demonstrated that ZbHaa1 is a ScHaa1 functional homologue by rescuing the acetic acid susceptibility phenotype of S. cerevisiae haa1Δ. The disruption of ZbHAA1 in Z. bailii IST302 and the expression of an extra ZbHAA1 copy confirmed ZbHAA1 as a determinant of acetic acid tolerance. ZbHaa1 was found to be required for acetic acid stress-induced transcriptional activation of Z. bailii genes homologous to ScHaa1-target genes. An evolutionary analysis of the Haa1 homologues identified in 28 Saccharomycetaceae species genome sequences, including Z bailii, was carried out using phylogenetic and gene neighbourhood approaches. Consistent with previous studies, this analysis revealed a group containing pre-whole genome duplication species Haa1/Cup2 single orthologues, including ZbHaa1, and two groups containing either Haa1 or Cup2 orthologues from post-whole genome duplication species. S. cerevisiae Cup2 (alias Ace1) is a transcription factor involved in response and tolerance to copper stress. Taken together, these observations led us to hypothesize and demonstrate that ZbHaa1 is also involved in copper-induced transcriptional regulation and copper tolerance. The transcription factor ZbHaa1 is required for adaptive response and tolerance to both acetic acid and copper stresses. The subfunctionalization of the single ancestral Haa1/Cup2 orthologue that originated Haa1 and Cup2 paralogues after whole genome duplication is proposed.

  13. Conductivity Probe

    Science.gov (United States)

    2008-01-01

    The Thermal and Electrical Conductivity Probe (TECP) for NASA's Phoenix Mars Lander took measurements in Martian soil and in the air. The needles on the end of the instrument were inserted into the Martian soil, allowing TECP to measure the propagation of both thermal and electrical energy. TECP also measured the humidity in the surrounding air. The needles on the probe are 15 millimeters (0.6 inch) long. The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  14. Aquatic Life Criteria - Copper

    Science.gov (United States)

    Documents pertain to Aquatic Life Ambient Water Quality criteria for Copper (2007 Freshwater, 2016 Estuarine/marine). These documents contain the safe levels of Copper in water that should protect to the majority of species.

  15. Antwerp Copper Plates

    DEFF Research Database (Denmark)

    Wadum, Jørgen

    1999-01-01

    In addition to presenting a short history of copper paintings, topics detail artists’ materials and techniques, as well as aspects of the copper industry, including mining, preparation and trade routes....

  16. Copper accumulation by stickleback nests containing spiggin.

    Science.gov (United States)

    Pinho, G L L; Martins, C M G; Barber, I

    2016-07-01

    The three-spined stickleback is a ubiquitous fish of marine, brackish and freshwater ecosystems across the Northern hemisphere that presents intermediate sensitivity to copper. Male sticklebacks display a range of elaborate reproductive behaviours that include nest construction. To build the nests, each male binds nesting material together using an endogenous glycoprotein nesting glue, known as 'spiggin'. Spiggin is a cysteine-rich protein and, therefore, potentially binds heavy metals present in the environment. The aim of this study was to investigate the capacity of stickleback nests to accumulate copper from environmental sources. Newly built nests, constructed by male fish from polyester threads in laboratory aquaria, were immersed in copper solutions ranging in concentration from 21.1-626.6 μg Cu L(-1). Bundles of polyester threads from aquaria without male fish were also immersed in the same copper solutions. After immersion, nests presented higher amounts of copper than the thread bundles, indicating a higher capacity of nests to bind this metal. A significant, positive correlation between the concentration of copper in the exposure solution and in the exposed nests was identified, but there was no such relationship for thread bundles. Since both spiggin synthesis and male courtship behaviour are under the control of circulating androgens, we predicted that males with high courtship scores would produce and secrete high levels of the spiggin protein. In the present study, nests built by high courtship score males accumulated more copper than those built by low courtship score males. Considering the potential of spiggin to bind metals, the positive relationship between fish courtship and spiggin secretion seems to explain the higher amount of copper on the nests from the fish showing high behaviour scores. Further work is now needed to determine the consequences of the copper binding potential of spiggin in stickleback nests for the health and survival of

  17. Mutant copper-zinc superoxide dismutase (SOD1) induces protein secretion pathway alterations and exosome release in astrocytes: implications for disease spreading and motor neuron pathology in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Basso, Manuela; Pozzi, Silvia; Tortarolo, Massimo; Fiordaliso, Fabio; Bisighini, Cinzia; Pasetto, Laura; Spaltro, Gabriella; Lidonnici, Dario; Gensano, Francesco; Battaglia, Elisa; Bendotti, Caterina; Bonetto, Valentina

    2013-05-31

    Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.

  18. Demystifying Controlling Copper Corrosion

    Science.gov (United States)

    The LCR systematically misses the highest health and corrosion risk sites for copper. Additionally, there are growing concerns for WWTP copper in sludges and discharge levels. There are many corrosion control differences between copper and lead. This talk explains the sometimes c...

  19. Pollution Probe.

    Science.gov (United States)

    Chant, Donald A.

    This book is written as a statement of concern about pollution by members of Pollution Probe, a citizens' anti-pollution group in Canada. Its purpose is to create public awareness and pressure for the eventual solution to pollution problems. The need for effective government policies to control the population explosion, conserve natural resources,…

  20. Probe specificity

    International Nuclear Information System (INIS)

    Laget, J.M.

    1986-11-01

    Specificity and complementarity of hadron and electron probes must be systematically developed to answer three questions currently asked in intermediate energy nuclear physics: what is nucleus structure at short distances, what is nature of short range correlations, what is three body force nature [fr

  1. Activation of chick tendon lysyl oxidase in response to dietary copper.

    Science.gov (United States)

    Rucker, R B; Rucker, B R; Mitchell, A E; Cui, C T; Clegg, M; Kosonen, T; Uriu-Adams, J Y; Tchaparian, E H; Fishman, M; Keen, C L

    1999-12-01

    Lysyl oxidase (EC 1.4.3.13), a cuproenzyme, can account for 10-30% of the copper present in connective tissue. Herein, we assess the extent to which tissue copper concentrations and lysyl oxidase activity are related because the functional activity of lysyl oxidase and the copper content of chick tendon are both related to dietary copper intake. Chicks (1-d old) were fed diets (basal copper concentration, 0.4 microg/g diet) to which copper was added from 0 to 16 microg/g diet. Liver and plasma copper levels tended to normalize in chickens that consumed from 1 to 4 microg copper/g of diet, whereas tendon copper concentrations suggested an unusual accumulation of copper in chickens that consumed 16 microg copper/g diet. The molecular weight of lysyl oxidase was also estimated using matrix-assisted laser desorption ionization/time-of-flight/mass spectrometry (MALDI/TOF/MS). A novel aspect of these measurements was estimation of protein mass directly from the surface of chick tendons and aortae. Whether copper deficiency (0 added copper) or copper supplementation (16 microg copper/g of diet) caused changes in the molecular weight of protein(s) in tendon corresponding to lysyl oxidase was addressed. The average molecular weight of the peak corresponding to lysyl oxidase in tendon and aorta from copper-deficient birds was 28,386 Da +/- 86, whereas the average molecular weight of corresponding protein in tendon from copper-supplemented birds was 28,639 Da +/- 122. We propose that the shift in molecular weight is due in part to copper binding and the formation of lysyl tyrosyl quinone, the cofactor at the active site of lysyl oxidase.

  2. A novel Schiff base derived from the gabapentin drug and copper (II) complex: Synthesis, characterization, interaction with DNA/protein and cytotoxic activity.

    Science.gov (United States)

    Shokohi-Pour, Zahra; Chiniforoshan, Hossein; Momtazi-Borojeni, Amir Abbas; Notash, Behrouz

    2016-09-01

    A novel Schiff base [C20H23NO3], has been prepared and characterized using FT-IR, UV-vis, (1)H NMR spectroscopy, elemental analysis and X-ray crystallography. A copper (II) complex [Cu(C20H22NO3)2]·H2O has also been synthesized and characterized. The new ligand and complex thus obtained were investigated by their interaction with calf thymus DNA and BSA using electronic absorption spectroscopy, fluorescence spectroscopy, and thermal denaturation. The intrinsic binding constants Kb of the ligand and Cu (II) complex, with CT-DNA obtained from UV-vis absorption studies were 1.53×10(4)M(-1) and 3.71×10(5)M(-1), respectively. Moreover the addition of the two compounds to CT-DNA (1:2) led to an increase of the melting temperature of DNA up to around 2.61°C for the ligand and 3.99°C for the Cu (II) complex. The ligand and Cu (II) complex bind to CT-DNA via a partial intercalative, as shown by the experimental data. In addition, the albumin interactions of the two compounds were studied by fluorescence quenching spectra, the results indicating that the binding mechanism is a static quenching process. The in vitro cytotoxicity of the two compounds on three different cancer cell lines was evaluated by MTT assay. The results showed that the copper complex exerted enhanced cytotoxicity compared with the Schiff base ligand; thereby, this complex clearly implies a positive synergistic effect. Furthermore, the copper complex showed a high, selective, and dose-dependent cytotoxicity against cancer cell lines. Copyright © 2016. Published by Elsevier B.V.

  3. Synthesis and crystal structure of binuclear copper(II) complex bridged by N-(2-hydroxyphenyl)-N'-[3-(diethylamino)propyl]oxamide: in vitro anticancer activity and reactivity toward DNA and protein.

    Science.gov (United States)

    Gao, Yang; Li, Yan-Tuan; Wu, Zhi-Yong; Yan, Cui-Wei

    2013-08-01

    A new oxamido-bridged bicopper(II) complex, [Cu2(pdpox)(bpy)(CH3OH)](ClO4), where H3pdpox and bpy stand for N-(2-hydroxyphenyl)-N'-[3-(diethylamino)propyl]oxamide and 2,2'-bipyridine, respectively, has been synthesized and characterized by elemental analyses, molar conductivity measurements, infrared and electronic spectra studies, and X-ray single crystal diffraction. In the crystal structure, the pdpox(3-) ligand bridges two copper(II) ions as cisoid conformation. The inner copper(II) ion has a {N3O} square-planar coordination geometry, while the exo- one is in a {N2O3} square-pyramidal environment. There are two sets of interpenetrating two-dimensional hydrogen bonding networks parallel to the planes (2 1 0) and (21¯0), respectively, to form a three-dimensional supramolecular structure. The bicopper(II) complex exhibits cytotoxic activity against the SMMC7721 and A549 cell lines. The reactivity toward herring sperm DNA and bovine serum albumin revealed that the bicopper(II) complex can interact with the DNA by intercalation mode, and the complex binds to protein BSA responsible for quenching of tryptophan fluorescence by static quenching mechanism. © 2013 Wiley Periodicals, Inc.

  4. Mechanisms of copper ion mediated Huntington's disease progression.

    Directory of Open Access Journals (Sweden)

    Jonathan H Fox

    2007-03-01

    Full Text Available Huntington's disease (HD is caused by a dominant polyglutamine expansion within the N-terminus of huntingtin protein and results in oxidative stress, energetic insufficiency and striatal degeneration. Copper and iron are increased in the striata of HD patients, but the role of these metals in HD pathogenesis is unknown. We found, using inductively-coupled-plasma mass spectroscopy, that elevations of copper and iron found in human HD brain are reiterated in the brains of affected HD transgenic mice. Increased brain copper correlated with decreased levels of the copper export protein, amyloid precursor protein. We hypothesized that increased amounts of copper bound to low affinity sites could contribute to pro-oxidant activities and neurodegeneration. We focused on two proteins: huntingtin, because of its centrality to HD, and lactate dehydrogenase (LDH, because of its documented sensitivity to copper, necessity for normoxic brain energy metabolism and evidence for altered lactate metabolism in HD brain. The first 171 amino acids of wild-type huntingtin, and its glutamine expanded mutant form, interacted with copper, but not iron. N171 reduced Cu(2+in vitro in a 1:1 copper:protein stoichiometry indicating that this fragment is very redox active. Further, copper promoted and metal chelation inhibited aggregation of cell-free huntingtin. We found decreased LDH activity, but not protein, and increased lactate levels in HD transgenic mouse brain. The LDH inhibitor oxamate resulted in neurodegeneration when delivered intra-striatially to healthy mice, indicating that LDH inhibition is relevant to neurodegeneration in HD. Our findings support a role of pro-oxidant copper-protein interactions in HD progression and offer a novel target for pharmacotherapeutics.

  5. Copper accumulation in senescent cells: Interplay between copper transporters and impaired autophagy

    Directory of Open Access Journals (Sweden)

    Shashank Masaldan

    2018-06-01

    Full Text Available Cellular senescence is characterized by irreversible growth arrest incurred through either replicative exhaustion or by pro-oncogenic cellular stressors (radioactivity, oxidative stress, oncogenic activation. The enrichment of senescent cells in tissues with age has been associated with tissue dyshomeostasis and age-related pathologies including cancers, neurodegenerative disorders (e.g. Alzheimer's, Parkinson's, etc. and metabolic disorders (e.g. diabetes. We identified copper accumulation as being a universal feature of senescent cells [mouse embryonic fibroblasts (MEF, human prostate epithelial cells and human diploid fibroblasts] in vitro. Elevated copper in senescent MEFs was accompanied by elevated levels of high-affinity copper uptake protein 1 (Ctr1, diminished levels of copper-transporting ATPase 1 (Atp7a (copper export and enhanced antioxidant defence reflected by elevated levels of glutathione (GSH, superoxide dismutase 1 (SOD1 and glutaredoxin 1 (Grx1. The levels of intracellular copper were further increased in senescent MEFs cultured in copper supplemented medium and in senescent Mottled Brindled (Mobr MEFs lacking functional Atp7a. Finally, we demonstrated that the restoration/preservation of autophagic-lysosomal degradation in senescent MEFs following rapamycin treatment correlated with attenuation of copper accumulation in these cells despite a further decrease in Atp7a levels. This study for the first time establishes a link between Atp7a and the autophagic-lysosomal pathway, and a requirement for both to effect efficient copper export. Such a connection between cellular autophagy and copper homeostasis is significant, as both have emerged as important facets of age-associated degenerative disease. Keywords: Senescence, Copper, Ageing, Homeostasis, Autophagy

  6. Transfer of Copper from an Amyloid to a Natural Copper-Carrier Peptide with a Specific Mediating Ligand.

    Science.gov (United States)

    Nguyen, Michel; Bijani, Christian; Martins, Nathalie; Meunier, Bernard; Robert, Anne

    2015-11-16

    The oxidative stress that arises from the catalytic reduction of dioxygen by Cu(II/I)-loaded amyloids is the major pathway for neuron death that occurs in Alzheimer's disease. In this work, we show that bis-8(aminoquinoline) ligands, copper(II) specific chelators, are able to catalytically extract Cu(II) from Cu-Aβ1-16 and then completely release Cu(I) in the presence of glutathione to provide a Cu(I)-glutathione complex, a biological intermediate that is able to deliver copper to apo forms of copper-protein complexes. These data demonstrate that bis-8(aminoquinolines) can perform the transfer of copper ions from the pathological Cu-amyloid complexes to regular copper-protein complexes. These copper-specific ligands assist GSH to recycle Cu(I) in an AD brain and consequently slow down oxidative damage that is due to copper dysregulation in Alzheimer's disease. Under the same conditions, we have shown that the copper complex of PBT2, a mono(8-hydroxyquinoline) previously used as a drug candidate, does not efficiently release copper in the presence of GSH. In addition, we report that GSH itself was unable to fully abstract copper ions from Cu-β-amyloid complexes. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Energetics of copper trafficking between the Atx1 metallochaperone and the intracellular copper transporter, Ccc2.

    Science.gov (United States)

    Huffman, D L; O'Halloran, T V

    2000-06-23

    The Atx1 metallochaperone protein is a cytoplasmic Cu(I) receptor that functions in intracellular copper trafficking pathways in plants, microbes, and humans. A key physiological partner of the Saccharomyces cerevisiae Atx1 is Ccc2, a cation transporting P-type ATPase located in secretory vesicles. Here, we show that Atx1 donates its metal ion cargo to the first N-terminal Atx1-like domain of Ccc2 in a direct and reversible manner. The thermodynamic gradient for metal transfer is shallow (K(exchange) = 1.4 +/- 0.2), establishing that vectorial delivery of copper by Atx1 is not based on a higher copper affinity of the target domain. Instead, Atx1 allows rapid metal transfer to its partner. This equilibrium is unaffected by a 50-fold excess of the Cu(I) competitor, glutathione, indicating that Atx1 also protects Cu(I) from nonspecific reactions. Mechanistically, we propose that a low activation barrier for transfer between partners results from complementary electrostatic forces that ultimately orient the metal-binding loops of Atx1 and Ccc2 for formation of copper-bridged intermediates. These thermodynamic and kinetic considerations suggest that copper trafficking proteins overcome the extraordinary copper chelation capacity of the eukaryotic cytoplasm by catalyzing the rate of copper transfer between physiological partners. In this sense, metallochaperones work like enzymes, carefully tailoring energetic barriers along specific reaction pathways but not others.

  8. EXPLORING MOLECULAR OXYGEN PATHWAYS IN HANSENULA POLYMORPHA COPPER-CONTAINING AMINE OXIDASE*

    Science.gov (United States)

    Johnson, Bryan J.; Cohen, Jordi; Welford, Richard W.; Pearson, Arwen R.; Schulten, Klaus; Klinman, Judith P.; Wilmot, Carrie M.

    2011-01-01

    The accessibility of large substrates to buried enzymatic active sites is dependent upon the utilization of proteinaceous channels. The necessity of these channels in the case of small substrates is questionable as diffusion through the protein matrix is often assumed. Copper amine oxidases (CAOs) contain a buried protein-derived quinone cofactor and a mononuclear copper center that catalyze the conversion of two substrates, primary amines and molecular oxygen, to aldehydes and hydrogen peroxide respectively. The nature of molecular oxygen migration to the active site in the enzyme from Hansenula polymorpha2 (HPAO) is explored using a combination of kinetic, X-ray crystallographic and computational approaches. A crystal structure of HPAO in complex with xenon gas, which serves as an experimental probe for molecular oxygen binding sites, reveals buried regions of the enzyme suitable for transient molecular oxygen occupation. Calculated O2 free energy maps using CAO crystal structures in the absence of xenon, correspond well with later experimentally observed xenon sites in these systems, and allow the visualization of O2 migration routes of differing probabilities within the protein matrix. Site-directed mutagenesis designed to block individual routes has little effect on overall kcat/Km[O2], supporting multiple dynamic pathways for molecular oxygen to reach the active site. PMID:17409383

  9. Effects of methionine chelate- or yeast proteinate-based supplement of copper, iron, manganese and zinc on broiler growth performance, their distribution in the tibia and excretion into the environment.

    Science.gov (United States)

    Singh, Abhay Kumar; Ghosh, Tapan Kumar; Haldar, Sudipto

    2015-04-01

    A straight-run flock of 1-day-old Cobb 400 chicks (n = 432) was distributed into four treatment groups (9 replicate pens in each group, 12 birds in a pen) for a 38-day feeding trial evaluating the effects of a methionine chelate (Met-TM)- or a yeast proteinate (Yeast-TM)-based supplement of copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn) on growth performance, bone criteria and some metabolic indices in commercial broiler chickens. The diets were either not supplemented with any trace elements at all (negative control, NC) or supplemented with an inorganic (sulphate) trace element premix (inorganic TM (ITM), 1 g/kg feed), the Met-TM (1 g/kg feed) and the Yeast-TM (0.5 g/kg feed). Body weight, feed conversion ratio and dressed meat yield at 38 days were better in the Yeast-TM-supplemented group as compared with the NC, ITM and Met-TM groups (p broilers with methionine chelates or yeast proteinate forms of Cu, Fe, Mn and Zn improved body weight and feed conversion ratio (FCR) and markedly reduced excretion of the said trace elements. The study revealed that it may be possible to improve broiler performance and reduce excretion of critical trace elements into the environment by complete replacement of inorganic trace minerals from their dietary regime and replacing the same with methionine chelate or yeast proteinate forms.

  10. Mixed-ligand copper(ii) Schiff base complexes: the role of the co-ligand in DNA binding, DNA cleavage, protein binding and cytotoxicity.

    Science.gov (United States)

    Lian, Wen-Jing; Wang, Xin-Tian; Xie, Cheng-Zhi; Tian, He; Song, Xue-Qing; Pan, He-Ting; Qiao, Xin; Xu, Jing-Yuan

    2016-05-31

    Four novel mononuclear Schiff base copper(ii) complexes, namely, [Cu(L)(OAc)]·H2O (), [Cu(HL)(C2O4)(EtOH)]·EtOH (), [Cu(L)(Bza)] () and [Cu(L)(Sal)] () (HL = 1-(((2-((2-hydroxypropyl)amino)ethyl)imino)methyl)naphthalene-2-ol), Bza = benzoic acid, Sal = salicylic acid), were synthesized and characterized by X-ray crystallography, elemental analysis and infrared spectroscopy. Single-crystal diffraction analysis revealed that all the complexes were mononuclear molecules, in which the Schiff base ligand exhibited different coordination modes and conformations. The N-HO and O-HO inter- and intramolecular hydrogen bonding interactions linked these molecules into multidimensional networks. Their interactions with calf thymus DNA (CT-DNA) were investigated by UV-visible and fluorescence spectrometry, as well as by viscosity measurements. The magnitude of the Kapp values of the four complexes was 10(5), indicating a moderate intercalative binding mode between the complexes and DNA. Electrophoresis results showed that all these complexes induced double strand breaks of pUC19 plasmid DNA in the presence of H2O2 through an oxidative pathway. In addition, the fluorescence spectrum of human serum albumin (HSA) with the complexes suggested that the quenching mechanism of HSA by the complexes was a static process. Moreover, the antiproliferative activity of the four complexes against HeLa (human cervical carcinoma) and HepG-2 (human liver hepatocellular carcinoma) cells evaluated by colorimetric cell proliferation assay and clonogenic assay revealed that all four complexes had improved cytotoxicity against cancer cells. Inspiringly, complex , with salicylic acid as the auxiliary ligand, displayed a stronger anticancer activity, suggesting that a synergistic effect of the Schiff base complex and the nonsteroidal anti-inflammatory drug may be involved in the cell killing process. The biological features of mixed-ligand copper(ii) Schiff base complexes and how acetic auxiliary

  11. Copper-dependent degradation of the Saccharomyces cerevisiae plasma membrane copper transporter Ctr1p in the apparent absence of endocytosis.

    OpenAIRE

    Ooi, C E; Rabinovich, E; Dancis, A; Bonifacino, J S; Klausner, R D

    1996-01-01

    The cell surface protein repertoire needs to be regulated in response to changes in the extracellular environment. In this study, we investigate protein turnover of the Saccharomyces cerevisiae plasma membrane copper transporter Ctr1p, in response to a change in extra-cellular copper levels. As Ctr1p mediates high affinity uptake of copper into the cell, modulation of its expression is expected to be involved in copper homeostasis. We demonstrate that Ctr1p is a stable protein when cells are ...

  12. The location of protein S8 and surrounding elements of 16S rRNA in the 70S ribosome from combined use of directed hydroxyl radical probing and X-ray crystallography.

    Science.gov (United States)

    Lancaster, L; Culver, G M; Yusupova, G Z; Cate, J H; Yusupov, M M; Noller, H F

    2000-05-01

    Ribosomal protein S8, which is essential for the assembly of the central domain of 16S rRNA, is one of the most thoroughly studied RNA-binding proteins. To map its surrounding RNA in the ribosome, we carried out directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to nine different positions on the surface of protein S8 in 70S ribosomes. Hydroxyl radical-induced cleavage was observed near the classical S8-binding site in the 620 stem, and flanking the other S8-footprinted regions of the central domain at the three-helix junction near position 650 and the 825 and 860 stems. In addition, cleavage near the 5' terminus of 16S rRNA, in the 300 region of its 5' domain, and in the 1070 region of its 3'-major domain provide information about the proximity to S8 of RNA elements not directly involved in its binding. These data, along with previous footprinting and crosslinking results, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-A map of the Thermus thermophilus 70S ribosome. The resulting model is in close agreement with the extensive body of data from previous studies using protein-protein and protein-RNA crosslinking, chemical and enzymatic footprinting, and genetics.

  13. Selection of fecal enterococci exhibiting tcrB-mediated copper resistance in pigs fed diets supplemented with copper.

    Science.gov (United States)

    Amachawadi, R G; Shelton, N W; Shi, X; Vinasco, J; Dritz, S S; Tokach, M D; Nelssen, J L; Scott, H M; Nagaraja, T G

    2011-08-15

    Copper, as copper sulfate, is increasingly used as an alternative to in-feed antibiotics for growth promotion in weaned piglets. Acquired copper resistance, conferred by a plasmid-borne, transferable copper resistance (tcrB) gene, has been reported in Enterococcus faecium and E. faecalis. A longitudinal field study was undertaken to determine the relationship between copper supplementation and the prevalence of tcrB-positive enterococci in piglets. The study was done with weaned piglets, housed in 10 pens with 6 piglets per pen, fed diets supplemented with a normal (16.5 ppm; control) or an elevated (125 ppm) level of copper. Fecal samples were randomly collected from three piglets per pen on days 0, 14, 28, and 42 and plated on M-Enterococcus agar, and three enterococcal isolates were obtained from each sample. The overall prevalence of tcrB-positive enterococci was 21.1% (38/180) in piglets fed elevated copper and 2.8% (5/180) in the control. Among the 43 tcrB-positive isolates, 35 were E. faecium and 8 were E. faecalis. The mean MICs of copper for tcrB-negative and tcrB-positive enterococci were 6.2 and 22.2 mM, respectively. The restriction digestion of the genomic DNA of E. faecium or E. faecalis with S1 nuclease yielded a band of ∼194-kbp size to which both tcrB and the erm(B) gene probes hybridized. A conjugation assay demonstrated cotransfer of tcrB and erm(B) genes between E. faecium and E. faecalis strains. The higher prevalence of tcrB-positive enterococci in piglets fed elevated copper compared to that in piglets fed normal copper suggests that supplementation of copper in swine diets selected for resistance.

  14. Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7

    International Nuclear Information System (INIS)

    Flores, J.; Sears, J.; Schael, I.P.; White, L.; Garcia, D.; Lanata, C.; Kapikian, A.Z.

    1990-01-01

    We have synthesized 32 P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies

  15. Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7

    Energy Technology Data Exchange (ETDEWEB)

    Flores, J.; Sears, J.; Schael, I.P.; White, L.; Garcia, D.; Lanata, C.; Kapikian, A.Z. (National Institutes of Health, Bethesda, MD (USA))

    1990-08-01

    We have synthesized {sup 32}P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies.

  16. Role of copper in the process of spermatogenesis

    Directory of Open Access Journals (Sweden)

    Mateusz Ogórek

    2017-08-01

    Full Text Available Copper (Cu is an essential trace element required for the normal development of living organisms. Due to its redox potential, copper is a cofactor in many enzymes responsible for important processes in cells. Copper deficiency has a significant influence on the reduction or the total eradication of copper-dependent enzymes in the body, thereby inhibiting cell life processes. On the other hand, copper is a very reactive element and in its free state, it can trigger the production of large amounts of free radicals, which will consequently lead to the damage of proteins and DNA. Because of those reasons, living organisms have developed precise mechanisms regulating the concentration of copper in cells. Copper also plays a very important role in male fertility. It is an essential element for the production of male gametes. The significant role of copper is also described in the processes of cell division – mitotic and meiotic. Copper-dependent enzymes such as ceruloplasmin, superoxide dismutase SOD1 and SOD3, group of metallothionein and cytochrome c oxidase are present at all stages of gametogenesis as well as in the somatic cells of the testis and in the somatic cells of epididymis. Substantial amounts of copper can also be found in liquids associated with sperm in the epididymis and prostate. Copper also affects the integral androgen distribution in terms of fertility on the line hypothalamic-pituitary-testis. Both copper increase and deficiency leads to a significant reduction in male fertility, which spans the entire spectrum of abnormalities at the sperm level, male gonad, production of hormones and distribution of micronutrients such as zinc and iron. Nowadays, the effects of copper on gametes production have become more important and are connected with the increasing levels of pollution with heavy metals in environment.

  17. Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis

    Directory of Open Access Journals (Sweden)

    Jonathon eTelianidis

    2013-08-01

    Full Text Available Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer’s, Parkinson’s and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-Type ATPases (copper-ATPases, ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration.

  18. Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode

    Czech Academy of Sciences Publication Activity Database

    Němcová, Kateřina; Šebest, Peter; Havran, Luděk; Orság, Petr; Fojta, Miroslav; Pivoňková, Hana

    2014-01-01

    Roč. 406, č. 24 (2014), s. 5843-5852 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GAP301/11/2076; GA AV ČR(CZ) IAA400040901 Institutional support: RVO:68081707 Keywords : Electrochemical analysis * Labeled probes * Osmium complex Subject RIV: BO - Biophysics Impact factor: 3.436, year: 2014

  19. Copper and silver halates

    CERN Document Server

    Woolley, EM; Salomon, M

    2013-01-01

    Copper and Silver Halates is the third in a series of four volumes on inorganic metal halates. This volume presents critical evaluations and compilations for halate solubilities of the Group II metals. The solubility data included in this volume are those for the five compounds, copper chlorate and iodate, and silver chlorate, bromate and iodate.

  20. Bioaccessibility and Solubility of Copper in Copper-Treated Lumber

    Science.gov (United States)

    Micronized copper (MC)-treated lumber is a recent replacement for Chromated Copper Arsenate (CCA) and Ammonium Copper (AC)-treated lumbers; though little is known about the potential risk of copper (Cu) exposure from incidental ingestion of MC-treated wood. The bioaccessibility o...

  1. Biochemical characterization of P-type copper ATPases

    Science.gov (United States)

    Inesi, Giuseppe; Pilankatta, Rajendra; Tadini-Buoninsegni, Francesco

    2014-01-01

    Copper ATPases, in analogy with other members of the P-ATPase superfamily, contain a catalytic headpiece including an aspartate residue reacting with ATP to form a phosphoenzyme intermediate, and transmembrane helices containing cation-binding sites [TMBS (transmembrane metal-binding sites)] for catalytic activation and cation translocation. Following phosphoenzyme formation by utilization of ATP, bound copper undergoes displacement from the TMBS to the lumenal membrane surface, with no H+ exchange. Although PII-type ATPases sustain active transport of alkali/alkali-earth ions (i.e. Na+, Ca2+) against electrochemical gradients across defined membranes, PIB-type ATPases transfer transition metal ions (i.e. Cu+) from delivery to acceptor proteins and, prominently in mammalian cells, undergo trafficking from/to various membrane compartments. A specific component of copper ATPases is the NMBD (N-terminal metal-binding domain), containing up to six copper-binding sites in mammalian (ATP7A and ATP7B) enzymes. Copper occupancy of NMBD sites and interaction with the ATPase headpiece are required for catalytic activation. Furthermore, in the presence of copper, the NMBD allows interaction with protein kinase D, yielding phosphorylation of serine residues, ATP7B trafficking and protection from proteasome degradation. A specific feature of ATP7A is glycosylation and stabilization on plasma membranes. Cisplatin, a platinum-containing anti-cancer drug, binds to copper sites of ATP7A and ATP7B, and undergoes vectorial displacement in analogy with copper. PMID:25242165

  2. Canine Copper-Associated Hepatitis

    NARCIS (Netherlands)

    Dirksen, Karen|info:eu-repo/dai/nl/412424428; Fieten, Hille|info:eu-repo/dai/nl/314112596

    2017-01-01

    Copper-associated hepatitis is recognized with increasing frequency in dogs. The disease is characterized by centrolobular hepatic copper accumulation, leading to hepatitis and eventually cirrhosis. The only way to establish the diagnosis is by histologic assessment of copper distribution and copper

  3. COPPER CABLE RECYCLING TECHNOLOGY

    International Nuclear Information System (INIS)

    Chelsea Hubbard

    2001-01-01

    The United States Department of Energy (DOE) continually seeks safer and more cost-effective technologies for use in deactivation and decommissioning (D and D) of nuclear facilities. The Deactivation and Decommissioning Focus Area (DDFA) of the DOE's Office of Science and Technology (OST) sponsors large-scale demonstration and deployment projects (LSDDPs). At these LSDDPs, developers and vendors of improved or innovative technologies showcase products that are potentially beneficial to the DOE's projects and to others in the D and D community. Benefits sought include decreased health and safety risks to personnel and the environment, increased productivity, and decreased costs of operation. The Idaho National Engineering and Environmental Laboratory (INEEL) generated a list of statements defining specific needs and problems where improved technology could be incorporated into ongoing D and D tasks. One such need is to reduce the volume of waste copper wire and cable generated by D and D. Deactivation and decommissioning activities of nuclear facilities generates hundreds of tons of contaminated copper cable, which are sent to radioactive waste disposal sites. The Copper Cable Recycling Technology separates the clean copper from contaminated insulation and dust materials in these cables. The recovered copper can then be reclaimed and, more importantly, landfill disposal volumes can be reduced. The existing baseline technology for disposing radioactively contaminated cables is to package the cables in wooden storage boxes and dispose of the cables in radioactive waste disposal sites. The Copper Cable Recycling Technology is applicable to facility decommissioning projects at many Department of Energy (DOE) nuclear facilities and commercial nuclear power plants undergoing decommissioning activities. The INEEL Copper Cable Recycling Technology Demonstration investigated the effectiveness and efficiency to recycle 13.5 tons of copper cable. To determine the effectiveness

  4. Crystal structures of E. coli laccase CueO at different copper concentrations

    International Nuclear Information System (INIS)

    Li Xu; Wei Zhiyi; Zhang Min; Peng Xiaohui; Yu Guangzhe; Teng Maikun; Gong Weimin

    2007-01-01

    CueO protein is a hypothetical bacterial laccase and a good laccase candidate for large scale industrial application. Four CueO crystal structures were determined at different copper concentrations. Low copper occupancy in apo-CueO and slow copper reconstitution process in CueO with exogenous copper were demonstrated. These observations well explain the copper dependence of CueO oxidase activity. Structural comparison between CueO and other three fungal laccase proteins indicates that Glu106 in CueO constitutes the primary counter-work for reconstitution of the trinuclear copper site. Mutation of Glu106 to a Phe enhanced CueO oxidation activity and supported this hypothesis. In addition, an extra α-helix from Leu351 to Gly378 covers substrate biding pocket of CueO and might compromises the electron transfer from substrate to type I copper

  5. Proximal Probes Facility

    Data.gov (United States)

    Federal Laboratory Consortium — The Proximal Probes Facility consists of laboratories for microscopy, spectroscopy, and probing of nanostructured materials and their functional properties. At the...

  6. Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays*

    Science.gov (United States)

    Yu, Xiaobo; Woolery, Andrew R.; Luong, Phi; Hao, Yi Heng; Grammel, Markus; Westcott, Nathan; Park, Jin; Wang, Jie; Bian, Xiaofang; Demirkan, Gokhan; Hang, Howard C.; Orth, Kim; LaBaer, Joshua

    2014-01-01

    AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications. PMID:25073739

  7. Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based detection of global pathogen-host AMPylation on self-assembled human protein microarrays.

    Science.gov (United States)

    Yu, Xiaobo; Woolery, Andrew R; Luong, Phi; Hao, Yi Heng; Grammel, Markus; Westcott, Nathan; Park, Jin; Wang, Jie; Bian, Xiaofang; Demirkan, Gokhan; Hang, Howard C; Orth, Kim; LaBaer, Joshua

    2014-11-01

    AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Reactive Chemical Probes: Beyond the Kinase Cysteinome.

    Science.gov (United States)

    Jones, Lyn H

    2018-04-12

    The reaction of small molecule chemical probes with proteins has been harnessed to develop covalent inhibitor drugs and protein profiling technologies. This Essay discusses some of the recent enhancements to the chemical biology toolkit that are enabling the study of previously unchartered areas of chemoproteomic space. An analysis of the kinome is used to illustrate the potential for these approaches to pursue new targets using reactive chemical probes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Nine New Fluorescent Probes

    Science.gov (United States)

    Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

    1989-06-01

    Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

  10. Micromachining with copper lasers

    Science.gov (United States)

    Knowles, Martyn R. H.; Bell, Andy; Foster-Turner, Gideon; Rutterford, Graham; Chudzicki, J.; Kearsley, Andrew J.

    1997-04-01

    In recent years the copper laser has undergone extensive development and has emerged as a leading and unique laser for micromachining. The copper laser is a high average power (10 - 250 W), high pulse repetition rate (2 - 32 kHz), visible laser (511 nm and 578 nm) that produces high peak power (typically 200 kW), short pulses (30 ns) and very good beam quality (diffraction limited). This unique set of laser parameters results in exceptional micro-machining in a wide variety of materials. Typical examples of the capabilities of the copper laser include the drilling of small holes (10 - 200 micrometer diameter) in materials as diverse as steel, ceramic, diamond and polyimide with micron precision and low taper (less than 1 degree) cutting and profiling of diamond. Application of the copper laser covers the electronic, aerospace, automotive, nuclear, medical and precision engineering industries.

  11. Sliding Friction of Copper

    National Research Council Canada - National Science Library

    Liu, Tung

    1963-01-01

    .... With less clean surfaces, the coefficient of friction obtained was about 0.4. Since the degree of cleanliness cannot be controlled quantitatively, the friction - load curve of sliding copper pairs in air exhibits a bifurcation characteristic...

  12. LEP copper accelerating cavities

    CERN Multimedia

    Laurent Guiraud

    1999-01-01

    These copper cavities were used to generate the radio frequency electric field that was used to accelerate electrons and positrons around the 27-km Large Electron-Positron (LEP) collider at CERN, which ran from 1989 to 2000. The copper cavities were gradually replaced from 1996 with new superconducting cavities allowing the collision energy to rise from 90 GeV to 200 GeV by mid-1999.

  13. Mycobacterium tuberculosis and Copper: A Newly Appreciated Defense against an Old Foe?

    Science.gov (United States)

    Darwin, K Heran

    2015-07-31

    Several independent studies have recently converged upon the conclusion that the human bacterial pathogen Mycobacterium tuberculosis encounters copper during infections. At least three independently regulated pathways respond to excess copper and are required for the full virulence of M. tuberculosis in animals. In this review, I will discuss the functions of the best-characterized copper-responsive proteins in M. tuberculosis, the potential sources of copper during an infection, and remaining questions about the interface between copper and tuberculosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Copper wire bonding

    CERN Document Server

    Chauhan, Preeti S; Zhong, ZhaoWei; Pecht, Michael G

    2014-01-01

    This critical volume provides an in-depth presentation of copper wire bonding technologies, processes and equipment, along with the economic benefits and risks.  Due to the increasing cost of materials used to make electronic components, the electronics industry has been rapidly moving from high cost gold to significantly lower cost copper as a wire bonding material.  However, copper wire bonding has several process and reliability concerns due to its material properties.  Copper Wire Bonding book lays out the challenges involved in replacing gold with copper as a wire bond material, and includes the bonding process changes—bond force, electric flame off, current and ultrasonic energy optimization, and bonding tools and equipment changes for first and second bond formation.  In addition, the bond–pad metallurgies and the use of bare and palladium-coated copper wires on aluminum are presented, and gold, nickel and palladium surface finishes are discussed.  The book also discusses best practices and re...

  15. New copper resistance determinants in the extremophile acidithiobacillus ferrooxidans: a quantitative proteomic analysis.

    Science.gov (United States)

    Almárcegui, Rodrigo J; Navarro, Claudio A; Paradela, Alberto; Albar, Juan Pablo; von Bernath, Diego; Jerez, Carlos A

    2014-02-07

    Acidithiobacillus ferrooxidans is an extremophilic bacterium used in biomining processes to recover metals. The presence in A. ferrooxidans ATCC 23270 of canonical copper resistance determinants does not entirely explain the extremely high copper concentrations this microorganism is able to stand, suggesting the existence of other efficient copper resistance mechanisms. New possible copper resistance determinants were searched by using 2D-PAGE, real time PCR (qRT-PCR) and quantitative proteomics with isotope-coded protein labeling (ICPL). A total of 594 proteins were identified of which 120 had altered levels in cells grown in the presence of copper. Of this group of proteins, 76 were up-regulated and 44 down-regulated. The up-regulation of RND-type Cus systems and different RND-type efflux pumps was observed in response to copper, suggesting that these proteins may be involved in copper resistance. An overexpression of most of the genes involved in histidine synthesis and several of those annotated as encoding for cysteine production was observed in the presence of copper, suggesting a possible direct role for these metal-binding amino acids in detoxification. Furthermore, the up-regulation of putative periplasmic disulfide isomerases was also seen in the presence of copper, suggesting that they restore copper-damaged disulfide bonds to allow cell survival. Finally, the down-regulation of the major outer membrane porin and some ionic transporters was seen in A. ferrooxidans grown in the presence of copper, indicating a general decrease in the influx of the metal and other cations into the cell. Thus, A. ferrooxidans most likely uses additional copper resistance strategies in which cell envelope proteins are key components. This knowledge will not only help to understand the mechanism of copper resistance in this extreme acidophile but may help also to select the best fit members of the biomining community to attain more efficient industrial metal leaching

  16. Comparison of the quantitative analysis performance between pulsed voltage atom probe and pulsed laser atom probe

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, J., E-mail: takahashi.3ct.jun@jp.nssmc.com [Advanced Technology Research Laboratories, Nippon Steel & Sumitomo Metal Corporation, 20-1 Shintomi, Futtsu-city, Chiba 293-8511 (Japan); Kawakami, K. [Advanced Technology Research Laboratories, Nippon Steel & Sumitomo Metal Corporation, 20-1 Shintomi, Futtsu-city, Chiba 293-8511 (Japan); Raabe, D. [Max-Planck Institut für Eisenforschung GmbH, Department for Microstructure Physics and Alloy Design, Max-Planck-Str. 1, 40237 Düsseldorf (Germany)

    2017-04-15

    Highlights: • Quantitative analysis in Fe-Cu alloy was investigated in voltage and laser atom probe. • In voltage-mode, apparent Cu concentration exceeded actual concentration at 20–40 K. • In laser-mode, the concentration never exceeded the actual concentration even at 20 K. • Detection loss was prevented due to the rise in tip surface temperature in laser-mode. • Preferential evaporation of solute Cu was reduced in laser-mode. - Abstract: The difference in quantitative analysis performance between the voltage-mode and laser-mode of a local electrode atom probe (LEAP3000X HR) was investigated using a Fe-Cu binary model alloy. Solute copper atoms in ferritic iron preferentially field evaporate because of their significantly lower evaporation field than the matrix iron, and thus, the apparent concentration of solute copper tends to be lower than the actual concentration. However, in voltage-mode, the apparent concentration was higher than the actual concentration at 40 K or less due to a detection loss of matrix iron, and the concentration decreased with increasing specimen temperature due to the preferential evaporation of solute copper. On the other hand, in laser-mode, the apparent concentration never exceeded the actual concentration, even at lower temperatures (20 K), and this mode showed better quantitative performance over a wide range of specimen temperatures. These results indicate that the pulsed laser atom probe prevents both detection loss and preferential evaporation under a wide range of measurement conditions.

  17. Biochemical characterization of the human copper transporter Ctr1.

    Science.gov (United States)

    Lee, Jaekwon; Peña, Maria Marjorette O; Nose, Yasuhiro; Thiele, Dennis J

    2002-02-08

    The trace metal copper is an essential cofactor for a number of biological processes including mitochondrial oxidative phosphorylation, free radical detoxification, neurotransmitter synthesis and maturation, and iron metabolism. Consequently, copper transport at the cell surface and the delivery of copper to intracellular proteins are critical events in normal physiology. Little is known about the molecules and biochemical mechanisms responsible for copper uptake at the plasma membrane in mammals. Here, we demonstrate that human Ctr1 (hCtr1) is a component of the copper transport machinery at the plasma membrane. hCtr1 transports copper with high affinity in a time-dependent and saturable manner and is metal-specific. hCtr1-mediated (64)Cu transport is an energy-independent process and is stimulated by extracellular acidic pH and high K(+) concentrations. hCtr1 exists as a homomultimer at the plasma membrane in mammalian cells. This is the first report on the biochemical characterization of the human copper transporter hCtr1, which is important for understanding mechanisms for mammalian copper transport at the plasma membrane.

  18. Cadmium versus copper toxicity: Insights from an integrated dissection of protein synthesis pathway in the digestive glands of mussel Mytilus galloprovincialis

    Energy Technology Data Exchange (ETDEWEB)

    Pytharopoulou, S.; Kournoutou, G.G. [Laboratory of Biochemistry, School of Medicine, University of Patras, 26504 Patras (Greece); Leotsinidis, M. [Laboratory of Public Health, School of Medicine, University of Patras, 26504 Patras (Greece); Georgiou, C.D. [Department of Biology, Section of Genetics, Cell Biology and Development, University of Patras, 26504 Patras (Greece); Kalpaxis, D.L., E-mail: dimkal@med.upatras.gr [Laboratory of Biochemistry, School of Medicine, University of Patras, 26504 Patras (Greece)

    2013-09-15

    Highlights: • Cu{sup 2+}-exposure of mussels results in genotoxicity, without affecting MTs production. •Cd{sup 2+}-exposure of mussels causes low genotoxicity, but induces MTs production. • Both metals induce oxidative stress in mussels, with Cd being the strongest inducer. • Translation is suppressed by both metals, mainly at the initiation and elongation steps. • MTs abrogate translational defects caused by Cd{sup 2+}, by trapping the toxic metal. -- Abstract: The main purpose of this study was to investigate the impact of metal-mediated stress on the protein-synthesis pathway in mussels. To this end, mussels (Mytilus galloprovincialis) underwent a 15 days exposure to 100 μg/L Cu{sup 2+} or Cd{sup 2+}. Both metals, in particular Cd{sup 2+}, accumulated in mussel digestive glands and generated a specific status of oxidative-stress. Exposure of mussels to each metal resulted in 40% decrease of the tRNA-aminoacylation efficiency, at the end of exposure. Cu{sup 2+} also caused a progressive loss in the capability of 40S-ribosomal subunits to form 48S pre-initiation complex, which reached 34% of the control at the end of exposure. Other steps of translation underwent less pronounced, but measurable damages. Mussels exposed to Cd{sup 2+} for 5 days presented a similar pattern of translational dysfunctions in digestive glands, but during the following days of exposure the ribosomal efficiency was gradually restored. Meanwhile, metallothionein levels significantly increased, suggesting that upon Cd{sup 2+}-mediated stress the protein-synthesizing activity was reorganized both quantitatively and qualitatively. Conclusively, Cd{sup 2+} and Cu{sup 2+} affect translation at several levels. However, the pattern of translational responses differs, largely depending on the capability of each metal to affect cytotoxic pathways in the tissues, such as induction of antioxidant defense and specific repair mechanisms.

  19. A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403.

    Science.gov (United States)

    Mancini, Stefano; Abicht, Helge K; Gonskikh, Yulia; Solioz, Marc

    2015-02-01

    Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD-yaiAB operon of Lactococcus lactis IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD-yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p-benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4-hydroxymuconic semialdehyde in an oxygen-consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD-yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments. © 2014 John Wiley & Sons Ltd.

  20. Direct patterning of probe proteins on an antifouling PLL-g-dextran coating for reducing the background signal of fluorescent immunoassays.

    Science.gov (United States)

    Egea, Amandine M C; Trévisiol, Emmanuelle; Vieu, Christophe

    2013-12-01

    The limit of detection of advanced immunoassays, biochips and micro/nano biodetection devices is impacted by the non-specific adsorption of target molecules at the sample surface. In this paper, we present a simple and versatile low cost method for generating active surfaces composed of antibodies arrays surrounded by an efficient anti-fouling layer, capable to decrease drastically the fluorescence background signal obtained after interaction with a solution to be analyzed. The technological process involves the direct micro-contact printing of the antibodies probe molecules on a pre-coated PLL-g-dextran thin layer obtained by contact printing using a flat PDMS stamp. Compared to other blocking strategies (ethanolamine blocking treatment, PLL-g-PEG incubation, PLL-g-dextran incubation, printing on a plasma-deposited PEO layer), our surface chemistry method is more efficient for reducing non-specific interactions responsible for a degraded signal/noise ratio.

  1. Mechanisms of Copper Ion Mediated Huntington's Disease Progression

    OpenAIRE

    Fox, Jonathan H.; Kama, Jibrin A.; Lieberman, Gregory; Chopra, Raman; Dorsey, Kate; Chopra, Vanita; Volitakis, Irene; Cherny, Robert A.; Bush, Ashley I.; Hersch, Steven

    2007-01-01

    Huntington's disease (HD) is caused by a dominant polyglutamine expansion within the N-terminus of huntingtin protein and results in oxidative stress, energetic insufficiency and striatal degeneration. Copper and iron are increased in the striata of HD patients, but the role of these metals in HD pathogenesis is unknown. We found, using inductively-coupled-plasma mass spectroscopy, that elevations of copper and iron found in human HD brain are reiterated in the brains of affected HD transgeni...

  2. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    International Nuclear Information System (INIS)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga; Luque, Daniel; Terrón, María C.; Calder, Lesley J.; Melero, José A.

    2014-01-01

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV F , we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV F at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy

  3. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    Energy Technology Data Exchange (ETDEWEB)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Luque, Daniel; Terrón, María C. [Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Calder, Lesley J. [National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA (United Kingdom); Melero, José A., E-mail: jmelero@isciii.es [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain)

    2014-07-15

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

  4. DNA and protein binding, double-strand DNA cleavage and cytotoxicity of mixed ligand copper(II) complexes of the antibacterial drug nalidixic acid.

    Science.gov (United States)

    Loganathan, Rangasamy; Ganeshpandian, Mani; Bhuvanesh, Nattamai S P; Palaniandavar, Mallayan; Muruganantham, Amsaveni; Ghosh, Swapan K; Riyasdeen, Anvarbatcha; Akbarsha, Mohammad Abdulkader

    2017-09-01

    The water soluble mixed ligand complexes [Cu(nal)(diimine)(H 2 O)](ClO 4 ) 1-4, where H(nal) is nalidixic acid and diimine is 2,2'-bipyridine (1), 1,10-phenanthroline (2), 5,6-dimethyl-1,10-phenanthroline (3), and 3,4,7,8-tetramethyl-1,10-phenanthroline (4), have been isolated. The coordination geometry around Cu(II) in 1 and that in the Density Functional Theory optimized structures of 1-4 has been assessed as square pyramidal. The trend in DNA binding constants (K b ) determined using absorption spectral titration (K b : 1, 0.79±0.1base pair. In contrast, 3 and 4 are involved in intimate hydrophobic interaction with DNA through the methyl substituents on phen ring, which is supported by viscosity and protein binding studies. DNA docking studies imply that 4 is involved preferentially in DNA major groove binding while 1-3 in minor groove binding and that all the complexes, upon removing the axially coordinated water molecule, bind in the major groove. Interestingly, 3 and 4 display prominent double-strand DNA cleavage while 1 and 2 effect only single-strand DNA cleavage in the absence of an activator. The complexes 3 and 4 show cytotoxicity higher than 1 and 2 against human breast cancer cell lines (MCF-7). The complex 4 induces apoptotic mode of cell death in cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Enzyme-mediated site-specific bioconjugation of metal complexes to proteins: sortase-mediated coupling of copper-64 to a single-chain antibody.

    Science.gov (United States)

    Paterson, Brett M; Alt, Karen; Jeffery, Charmaine M; Price, Roger I; Jagdale, Shweta; Rigby, Sheena; Williams, Charlotte C; Peter, Karlheinz; Hagemeyer, Christoph E; Donnelly, Paul S

    2014-06-10

    The enzyme-mediated site-specific bioconjugation of a radioactive metal complex to a single-chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single-chain variable fragment. The antibody fragment scFv(anti-LIBS) targets ligand-induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron-emitting isotope (64)Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site-specific enzyme-mediated conjugation and should be broadly applicable to other metal complexes and proteins. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. A Perspective on the Maillard Reaction and the Analysis of Protein Glycation by Mass Spectrometry: Probing the Pathogenesis of Chronic Disease

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qibin; Ames, Jennifer M.; Smith, Richard D.; Baynes, John; Metz, Thomas O.

    2008-12-18

    The Maillard reaction, starting from the glycation of protein and progressing to the formation of advanced glycation end-products (AGEs), is implicated in the development of complications of diabetes mellitus, as well as in the pathogenesis of cardiovascular, renal, and neurodegenerative diseases. In this perspective review, we provide on overview on the relevance of the Maillard reaction in the pathogenesis of chronic disease and discuss traditional approaches and recent developments in the analysis of glycated proteins by mass spectrometry. We propose that proteomics approaches, particularly bottom-up proteomics, will play a significant role in analyses of clinical samples leading to the identification of new markers of disease development and progression.

  7. Copper and copper-nickel alloys as zebra mussel antifoulants

    Energy Technology Data Exchange (ETDEWEB)

    Dormon, J.M.; Cottrell, C.M.; Allen, D.G.; Ackerman, J.D.; Spelt, J.K. [Univ. of Toronto, Ontario (Canada)

    1996-04-01

    Copper has been used in the marine environment for decades as cladding on ships and pipes to prevent biofouling by marine mussels (Mytilus edulis L.). This motivated the present investigation into the possibility of using copper to prevent biofouling in freshwater by both zebra mussels and quagga mussels (Dreissena polymorpha and D. bugensis collectively referred to as zebra mussels). Copper and copper alloy sheet proved to be highly effective in preventing biofouling by zebra mussels over a three-year period. Further studies were conducted with copper and copper-nickel mesh (lattice of expanded metal) and screen (woven wire with a smaller hole size), which reduced the amount of copper used. Copper screen was also found to be strongly biofouling-resistant with respect to zebra mussels, while copper mesh reduced zebra mussel biofouling in comparison to controls, but did not prevent it entirely. Preliminary investigations into the mechanism of copper antifouling, using galvanic couples, indicated that the release of copper ions from the surface of the exposed metal into the surrounding water is directly or indirectly responsible for the biofouling resistance of copper.

  8. Electrical conduction in composites containing copper core-copper ...

    Indian Academy of Sciences (India)

    Composites of nanometre-sized copper core-copper oxide shell with diameters in the range 6.1 to 7.3 nm dispersed in a silica gel were synthesised by a technique comprising reduction followed by oxidation of a suitably chosen precursor gel. The hot pressed gel powders mixed with nanometre-sized copper particles ...

  9. [An improved method of preparing protein and peptide probes in mass spectrometry with ionization of division fragments by californium-252 (TOF-PDMS)].

    Science.gov (United States)

    Chivanov, V D; Zubarev, R A; Aksenov, S A; Bordunova, O G; Eremenko, V I; Kabanets, V M; Tatarinova, V I; Mishnev, A K; Kuraev, V V; Knysh, A N; Eremenko, I A

    1996-08-01

    The addition of organic acids (picric, oxalic, citric, or tartaric) to peptide and protein samples was found to significantly increase the yield of their quasi-molecular ions (QMI) in time-of-flight 252Cf plasma desorption mass spectrometry. The yield of the ions depended on the pKa of the acid added.

  10. Hydrogen bond strength in membrane proteins probed by time-resolved 1 H-detected solid-state NMR and MD simulations

    NARCIS (Netherlands)

    Medeiros-silva, João; Jekhmane, Miranda; Baldus, Marc; Weingarth, Markus

    2017-01-01

    1H-detected solid-state NMR in combination with 1H/2D exchange steps allows for the direct identification of very strong hydrogen bonds in membrane proteins. On the example of the membrane-embedded potassium channel KcsA, we quantify the longevity of such very strong hydrogen bonds by combining

  11. Fluorinated Vitamin B12 Analogs Are Cofactors of Corrinoid-Dependent Enzymes: a 19F-Labeled Nuclear Magnetic Resonance Probe for Identifying Corrinoid-Protein Interactions

    Science.gov (United States)

    Stupperich, Erhard; Eisinger, Hans-Jürgen; Kerssebaum, Rainer; Nexø, Ebba

    1993-01-01

    The homoacetogenic bacterium Sporomusa ovata synthesized the vitamin B12 analog phenolyl cobamide or 4-fluorophenolyl cobamide when the methanol medium of growing cells was supplemented with 10 mM phenol or 5 mM 4-fluorophenol. Phenol and, presumably, 4-fluorophenol were specifically incorporated into these cobamides, since phenol was not metabolized significantly into amino acids or into acetic acid, the product of the catabolism. The phenol-containing cobamides contributed up to 90% of the protein-bound cobamides of the 1,300 to 1,900 nmol of corrinoid per g of dry cell material formed. Fluorine-19 nuclear magnetic resonance spectroscopy of 4-fluorophenolyl cobamide exhibited a resonance near 30 ppm. An additional signal emerged at 25 ppm when 4-fluorophenolyl cobamide was investigated as the cofactor of a corrinoid-dependent protein. The two resonances indicated distinct cofactor arrangements within the protein's active site. A 5-ppm high-field shift change suggested van der Waal's interactions between the fluorinated nucleotide of the cofactor and adjacent amino acid residues of the enzyme. Similarly, Propionibacterium freudenreichii and Methanobacterium thermoautotrophicum synthesized 5-fluorobenzimidazolyl cobamide. The human corrinoid binders intrinsic factor, transcobalamin, and haptocorrin recognized this corrinoid like vitamin B12. Hence, it is possible to use 19F-labeled nuclear magnetic resonance spectroscopy for analyses of protein-bound cobamides. PMID:16348877

  12. Alkylsulfonates as probes of uncoupling protein transport mechanism. Ion pair transport demonstrates that direct H(+) translocation by UCP1 is not necessary for uncoupling

    Czech Academy of Sciences Publication Activity Database

    Jabůrek, M.; Vařecha, M.; Ježek, Petr; Garlid, K. D.

    2001-01-01

    Roč. 276, č. 34 (2001), s. 31897-31905 ISSN 0021-9258 R&D Projects: GA AV ČR IAA5011106 Grant - others:NIH(US) DK56273 Institutional research plan: CEZ:AV0Z5011922 Keywords : mitochondrial uncoupling proteins * alkylsulfonates * ion pair transport Subject RIV: CE - Biochemistry Impact factor: 7.258, year: 2001

  13. Probing the Mechanism of pH-Induced Large-Scale Conformational Changes in Dengue Virus Envelope Protein Using Atomistic Simulations

    Science.gov (United States)

    Prakash, Meher K.; Barducci, Alessandro; Parrinello, Michele

    2010-01-01

    Abstract One of the key steps in the infection of the cell by dengue virus is a pH-induced conformational change of the viral envelope proteins. These envelope proteins undergo a rearrangement from a dimer to a trimer, with large conformational changes in the monomeric unit. In this article, metadynamics simulations were used to enable us to understand the mechanism of these large-scale changes in the monomer. By using all-atom, explicit solvent simulations of the monomers, the stability of the protein structure is studied under low and high pH conditions. Free energy profiles obtained along appropriate collective coordinates demonstrate that pH affects the domain interface in both the conformations of E monomer, stabilizing one and destabilizing the other. These simulations suggest a mechanism with an intermediate detached state between the two monomeric structures. Using further analysis, we comment on the key residue interactions responsible for the instability and the pH-sensing role of a histidine that could not otherwise be studied experimentally. The insights gained from this study and methodology can be extended for studying similar mechanisms in the E proteins of the other members of class II flavivirus family. PMID:20643078

  14. Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer.

    Science.gov (United States)

    Wollman, Adam J M; Miller, Helen; Zhou, Zhaokun; Leake, Mark C

    2015-04-01

    DNA-interacting proteins have roles in multiple processes, many operating as molecular machines which undergo dynamic meta-stable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner, fast enough to sample the molecular transitions across the free-energy landscape. Progress has been made over the past decade in utilizing cutting-edge tools of the physical sciences to address challenging biological questions concerning the function and modes of action of several different proteins which bind to DNA. These physiologically relevant assays are technically challenging but can be complemented by powerful and often more tractable in vitro experiments which confer advantages of the chemical environment with enhanced detection signal-to-noise of molecular signatures and transition events. In the present paper, we discuss a range of techniques we have developed to monitor DNA-protein interactions in vivo, in vitro and in silico. These include bespoke single-molecule fluorescence microscopy techniques to elucidate the architecture and dynamics of the bacterial replisome and the structural maintenance of bacterial chromosomes, as well as new computational tools to extract single-molecule molecular signatures from live cells to monitor stoichiometry, spatial localization and mobility in living cells. We also discuss recent developments from our laboratory made in vitro, complementing these in vivo studies, which combine optical and magnetic tweezers to manipulate and image single molecules of DNA, with and without bound protein, in a new super-resolution fluorescence microscope.

  15. THE ORNL ATOM PROBE

    OpenAIRE

    Miller, M.

    1986-01-01

    The ORNL Atom Probe is a microanalytical tool for studies in materials science. The instrument is a combination of a customized version of the vacuum system of the VG FIM-100 atom probe, an ORNL-designed microcomputer-controlled digital timing system, and a double curved CEMA Imaging Atom Probe detector. The atom probe combines four instruments into one - namely a field ion microscope, an energy compensated time-of-flight mass spectrometer, an imaging atom probe, and a pulsed laser atom probe.

  16. Mobile Game Probes

    DEFF Research Database (Denmark)

    Borup Lynggaard, Aviaja

    2006-01-01

    This paper will examine how probes can be useful for game designers in the preliminary phases of a design process. The work is based upon a case study concerning pervasive mobile phone games where Mobile Game Probes have emerged from the project. The new probes are aimed towards a specific target...... group and the goal is to specify the probes so they will cover the most relevant areas for our project. The Mobile Game Probes generated many interesting results and new issues occurred, since the probes came to be dynamic and favorable for the process in new ways....

  17. A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria.

    Science.gov (United States)

    Karunakaran, R; Mauchline, T H; Hosie, A H F; Poole, P S

    2005-10-01

    A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfpmut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT.3, dsRedT.4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA(p)) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.

  18. Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

    Science.gov (United States)

    Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L.; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

    2015-12-01

    Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

  19. Proteomic and physiological responses of Kineococcus radiotolerans to copper.

    Directory of Open Access Journals (Sweden)

    Christopher E Bagwell

    Full Text Available Copper is a highly reactive, toxic metal; consequently, transport of this metal within the cell is tightly regulated. Intriguingly, the actinobacterium Kineococcus radiotolerans has been shown to not only accumulate soluble copper to high levels within the cytoplasm, but the phenotype also correlated with enhanced cell growth during chronic exposure to ionizing radiation. This study offers a first glimpse into the physiological and proteomic responses of K. radiotolerans to copper at increasing concentration and distinct growth phases. Aerobic growth rates and biomass yields were similar over a range of Cu(II concentrations (0-1.5 mM in complex medium. Copper uptake coincided with active cell growth and intracellular accumulation was positively correlated with Cu(II concentration in the growth medium (R(2=0.7. Approximately 40% of protein coding ORFs on the K. radiotolerans genome were differentially expressed in response to the copper treatments imposed. Copper accumulation coincided with increased abundance of proteins involved in oxidative stress and defense, DNA stabilization and repair, and protein turnover. Interestingly, the specific activity of superoxide dismutase was repressed by low to moderate concentrations of copper during exponential growth, and activity was unresponsive to perturbation with paraquot. The biochemical response pathways invoked by sub-lethal copper concentrations are exceptionally complex; though integral cellular functions are preserved, in part, through the coordination of defense enzymes, chaperones, antioxidants and protective osmolytes that likely help maintain cellular redox. This study extends our understanding of the ecology and physiology of this unique actinobacterium that could potentially inspire new biotechnologies in metal recovery and sequestration, and environmental restoration.

  20. Proteomic and physiological responses of Kineococcus radiotolerans to copper.

    Science.gov (United States)

    Bagwell, Christopher E; Hixson, Kim K; Milliken, Charles E; Lopez-Ferrer, Daniel; Weitz, Karl K

    2010-08-26

    Copper is a highly reactive, toxic metal; consequently, transport of this metal within the cell is tightly regulated. Intriguingly, the actinobacterium Kineococcus radiotolerans has been shown to not only accumulate soluble copper to high levels within the cytoplasm, but the phenotype also correlated with enhanced cell growth during chronic exposure to ionizing radiation. This study offers a first glimpse into the physiological and proteomic responses of K. radiotolerans to copper at increasing concentration and distinct growth phases. Aerobic growth rates and biomass yields were similar over a range of Cu(II) concentrations (0-1.5 mM) in complex medium. Copper uptake coincided with active cell growth and intracellular accumulation was positively correlated with Cu(II) concentration in the growth medium (R(2)=0.7). Approximately 40% of protein coding ORFs on the K. radiotolerans genome were differentially expressed in response to the copper treatments imposed. Copper accumulation coincided with increased abundance of proteins involved in oxidative stress and defense, DNA stabilization and repair, and protein turnover. Interestingly, the specific activity of superoxide dismutase was repressed by low to moderate concentrations of copper during exponential growth, and activity was unresponsive to perturbation with paraquot. The biochemical response pathways invoked by sub-lethal copper concentrations are exceptionally complex; though integral cellular functions are preserved, in part, through the coordination of defense enzymes, chaperones, antioxidants and protective osmolytes that likely help maintain cellular redox. This study extends our understanding of the ecology and physiology of this unique actinobacterium that could potentially inspire new biotechnologies in metal recovery and sequestration, and environmental restoration.

  1. Activation Mechanism of the Streptomyces Tyrosinase Assisted by the Caddie Protein.

    Science.gov (United States)

    Matoba, Yasuyuki; Kihara, Shogo; Muraki, Yoshimi; Bando, Naohiko; Yoshitsu, Hironari; Kuroda, Teruo; Sakaguchi, Miyuki; Kayama, Kure'e; Tai, Hulin; Hirota, Shun; Ogura, Takashi; Sugiyama, Masanori

    2017-10-17

    Tyrosinase (EC 1.14.18.1), which possesses two copper ions at the active center, catalyzes a rate-limiting reaction of melanogenesis, that is, the conversion of a phenol to the corresponding ortho-quinone. The enzyme from the genus Streptomyces is generated as a complex with a "caddie" protein that assists the transport of two copper ions into the active center. In this complex, the Tyr 98 residue in the caddie protein was found to be accommodated in the pocket of the active center of tyrosinase, probably in a manner similar to that of l-tyrosine as a genuine substrate of tyrosinase. Under physiological conditions, the addition of the copper ion to the complex releases tyrosinase from the complex, in accordance with the aggregation of the caddie protein. The release of the copper-bound tyrosinase was found to be accelerated by adding reducing agents under aerobic conditions. Mass spectroscopic analysis indicated that the Tyr 98 residue was converted to a reactive quinone, and resonance Raman spectroscopic analysis indicated that the conversion occurred through the formations of μ-η 2 :η 2 -peroxo-dicopper(II) and Cu(II)-semiquinone. Electron paramagnetic resonance analysis under anaerobic conditions and Fourier transform infrared spectroscopic analysis using CO as a structural probe under anaerobic conditions indicated that the copper transportation process to the active center is a reversible event in the tyrosinase/caddie complex. Aggregation of the caddie protein, which is triggered by the conversion of the Tyr 98 residue to dopaquinone, may ensure the generation of fully activated tyrosinase.

  2. Brazing copper to dispersion-strengthened copper

    Energy Technology Data Exchange (ETDEWEB)

    Ryding, D.G.; Allen, D.; Lee, R.

    1996-08-01

    The Advanced Photon Source (APS) is a state-of-the-art synchrotron light source that will produce intense x-ray beams, which will allow the study of smaller samples and faster reactions and processes at a greater level of detail that has been possible to date. The beam is produced by using third-generation insertion devices in a 7 GeV electron/positron storage ring that is 1100 meters in circumference. The heat load from these intense high power devices is very high and certain components must sustain total heat loads of 3 to 15 kW and heat fluxes of 30 W/mm{sup 2}. Because the beams will cycle on and off many times, thermal shock and fatigue will be a problem. High heat flux impinging on a small area causes a large thermal gradient that results in high stress. GlidCop{reg_sign}, a dispersion strengthened copper, is the desired material because of its high thermal conductivity and superior mechanical properties as compared to copper and its alloys. GlidCop is not amenable to joining by fusion welding, and brazing requires diligence because of high diffusivity. Brazing procedures were developed using optical and scanning electron microscopy.

  3. Using Xas And Sxrf to Study Copper in Wilson Disease at the Molecular And Tissue Level

    Energy Technology Data Exchange (ETDEWEB)

    Ralle, M.; Blackburn, N.J.; Lutsenko, S.

    2009-06-05

    Wilson disease (WD) is a genetic disorder of copper metabolism associated with severe hepatic, neurological, and psychiatric abnormalities. In WD, the billiary copper excretion is impaired and copper accumulates in tissues, particularly in the liver and the brain. The affected gene, ATP7B, encodes the copper transporting ATPase, Wilson disease protein (WNDP). WNDP has six copper binding sites in the N-terminal portion of the molecule. Each site includes the conserved amino acid sequence MXCXXC, and binds 1 Cu(I) through its 2 cysteine residues. We performed X-ray absorption studies at the Cu K{sub {alpha}}-edge on the recombinant N-terminal domain of WNDP (N-WNDP). Copper was bound to N-WNDP either in vivo or in vitro in the presence of different reducing agents. We found that in N-WNDP copper is predominantly coordinated in a linear fashion by two cysteines, with the appearance of a Cu-Cu interaction when all metal binding sites are filled. Increasing amounts of reducing agents containing sulfide or phosphine groups led to binding of the exogenous ligands to copper thereby increasing the coordination number of copper from two to three. To better understand the role of copper in WD, we utilized livers of the 6-weeks-old Atp7b-/- mice (an animal model for WD) in which the copper concentration was 10--20-fold higher compared to that of the control mice. The distribution of copper in hepatocytes was evaluated by synchrotron based X-ray fluorescence microprobe (SXRF). We demonstrate that we can prepare liver slices that retain copper and can detect copper with subcellular resolution. On the same sections {mu}-XANES (spot size: 5 micron) was used to determine the oxidation state of copper.

  4. Using XAS and SXRF to Study Copper in Wilson Disease at the Molecular and Tissue Level

    Science.gov (United States)

    Ralle, Martina; Blackburn, Ninian J.; Lutsenko, Svetlana

    2007-02-01

    Wilson disease (WD) is a genetic disorder of copper metabolism associated with severe hepatic, neurological, and psychiatric abnormalities. In WD, the billiary copper excretion is impaired and copper accumulates in tissues, particularly in the liver and the brain. The affected gene, ATP7B, encodes the copper transporting ATPase, Wilson disease protein (WNDP). WNDP has six copper binding sites in the N-terminal portion of the molecule. Each site includes the conserved amino acid sequence MXCXXC, and binds 1 Cu(I) through its 2 cysteine residues. We performed X-ray absorption studies at the Cu Kα-edge on the recombinant N-terminal domain of WNDP (N-WNDP). Copper was bound to N-WNDP either in vivo or in vitro in the presence of different reducing agents. We found that in N-WNDP copper is predominantly coordinated in a linear fashion by two cysteines, with the appearance of a Cu-Cu interaction when all metal binding sites are filled. Increasing amounts of reducing agents containing sulfide or phosphine groups led to binding of the exogenous ligands to copper thereby increasing the coordination number of copper from two to three. To better understand the role of copper in WD, we utilized livers of the 6-weeks-old Atp7b-/- mice (an animal model for WD) in which the copper concentration was 10-20-fold higher compared to that of the control mice. The distribution of copper in hepatocytes was evaluated by synchrotron based X-ray fluorescence microprobe (SXRF). We demonstrate that we can prepare liver slices that retain copper and can detect copper with subcellular resolution. On the same sections μ-XANES (spot size: 5 micron) was used to determine the oxidation state of copper.

  5. The Effect of Orthophosphate as a Copper Corrosion Inhibitor in High Alkalinity Drinking Water Systems

    Science.gov (United States)

    2007-03-01

    in the near future. 7 2.2. Human Health Concerns In the human body, copper is a component of proteins that perform a range of functions...Studies have shown that on average, daily copper intake is 3200 μg. Copper comes from foods such as shellfish, meat, eggs, vegetables , nuts...dehydration synthesis, have a tendency to hydrolyze to orthophosphate in the presence of water, therefore altering their corrosion control mechanism (Cantor

  6. Micro determination of plasma and erythrocyte copper by atomic absorption spectrophotometry.

    Science.gov (United States)

    Blomfield, J; Macmahon, R A

    1969-03-01

    The free and total plasma copper and total erythrocyte copper levels have been determined by simple, yet sensitive and highly specific methods, using atomic absorption spectrophotometry. For total copper determination, the copper was split from its protein combination in plasma or red cells by the action of hydrochloric acid at room temperature. The liberated copper was chelated by ammonium pyrrolidine dithiocarbamate and extracted into n-butyl acetate by shaking and the organic extract was aspirated into the atomic absorption spectrophotometer flame. The entire procedure was carried out in polypropylene centrifuge tubes, capped during shaking. For the free plasma copper measurement the hydrochloric acid step was omitted. Removal of the plasma or erythrocyte proteins was found to be unnecessary, and, in addition, the presence of trichloracetic acid caused an appreciable lowering of absorption. Using a double-beam atomic absorption spectrophotometer and scale expansion x 10, micro methods have been derived for determining the total copper of plasma or erythrocytes with 0.1 ml of sample, and the free copper of plasma with 0.5 ml. The macro plasma copper method requires 2 ml of plasma and is suitable for use with single-beam atomic absorption spectrophotometers. With blood from 50 blood donors, normal ranges of plasma and erythrocyte copper have been determined.

  7. Coping with copper

    DEFF Research Database (Denmark)

    Nunes, Ines Marques; Jacquiod, Samuel Jehan Auguste; Brejnrod, Asker Daniel

    2016-01-01

    Copper has been intensively used in industry and agriculture since mid-18(th) century and is currently accumulating in soils. We investigated the diversity of potential active bacteria by 16S rRNA gene transcript amplicon sequencing in a temperate grassland soil subjected to century-long exposure...

  8. and copper(II)

    Indian Academy of Sciences (India)

    Unknown

    characterization of several imidazolate-bridged binuclear copper(II) complexes have been reported 1–17. ... of the desired complex formed were collected, washed with ethanol and dried in vacuo at room temperature. .... 16. Sigel H (ed.) 1981 Metal ions in biological system (New York: Marcel Dekker) vol 13, p. 259. 17.

  9. Role of copper in photochemical damage to hair.

    Science.gov (United States)

    Marsh, J M; Iveson, R; Flagler, M J; Davis, M G; Newland, A B; Greis, K D; Sun, Y; Chaudhary, T; Aistrup, E R

    2014-02-01

    The objective of this work was to identify whether low levels of redox metals such as copper will accelerate damage to hair on exposure to UV irradiation and whether this damage can be prevented. The methods used were proteomics to measure the protein damage via protein loss after different periods of exposure and mass spectroscopy methods to identify specific marker peptides that are specifically created by this type of damage. In this work, we have developed new insights into the mechanism of UV damage using these proteomic methods. A marker fragment in the hair protein loss extract was identified (m/z = 1279) that is unique to UV exposure and increases with time of UV exposure. We have also identified for the first time in hair the role of exogenous copper in increasing UV damage both in terms of total protein degradation and also increased formation of the marker fragment and proposed a mechanism of action. It has been demonstrated that shampoo treatment containing a chelant such as N,N'-ethylenediamine disuccinic acid (EDDS) reduced copper accumulation in hair. This work provides evidence for the role of copper in UV-induced damage to hair and strategies to reduce copper levels in hair using a chelant such as EDDS. © 2013 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  10. Probing the functional impact of sequence variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts.

    Directory of Open Access Journals (Sweden)

    Maher A Noureddine

    2009-05-01

    Full Text Available The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs. Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation -- including polymorphisms -- and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

  11. The interplay between transient a-helix formation and side chain rotamer distributions in disordered proteins probed by methyl chemical shifts

    DEFF Research Database (Denmark)

    Kjærgaard, Magnus; Iesmantavicius, Vytautas; Poulsen, Flemming M

    2011-01-01

    and retinoid receptors (ACTR). We find that small differences in the methyl carbon chemical shifts due to the ¿-gauche effect may provide information about the side chain rotamer distributions. However, the effects of neighboring residues on the methyl group chemical shifts obscure the direct observation...... a quantitative analysis of the ensemble of ¿(2)-angles of especially leucine residues in disordered proteins. The changes in the rotamer distributions upon denaturation correlate to the changes upon helix induction by the co-solvent trifluoroethanol, suggesting that the side chain conformers are directly...

  12. Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl tranferase. Application in electrochemical DNA hybridization and protein-DNA binding assays

    Czech Academy of Sciences Publication Activity Database

    Horáková Brázdilová, Petra; Macíčková-Cahová, Hana; Pivoňková, Hana; Špaček, Jan; Havran, Luděk; Hocek, Michal; Fojta, Miroslav

    2011-01-01

    Roč. 9, č. 5 (2011), s. 1366-1371 ISSN 1477-0520 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk(CZ) LC512; GA AV ČR(CZ) IAA400040901 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702; CEZ:AV0Z40550506 Keywords : DNA tail-labelling * protein-DNA binding * DNA hybridization Subject RIV: BO - Biophysics Impact factor: 3.696, year: 2011

  13. Comparison of the quantitative analysis performance between pulsed voltage atom probe and pulsed laser atom probe.

    Science.gov (United States)

    Takahashi, J; Kawakami, K; Raabe, D

    2017-04-01

    The difference in quantitative analysis performance between the voltage-mode and laser-mode of a local electrode atom probe (LEAP3000X HR) was investigated using a Fe-Cu binary model alloy. Solute copper atoms in ferritic iron preferentially field evaporate because of their significantly lower evaporation field than the matrix iron, and thus, the apparent concentration of solute copper tends to be lower than the actual concentration. However, in voltage-mode, the apparent concentration was higher than the actual concentration at 40K or less due to a detection loss of matrix iron, and the concentration decreased with increasing specimen temperature due to the preferential evaporation of solute copper. On the other hand, in laser-mode, the apparent concentration never exceeded the actual concentration, even at lower temperatures (20K), and this mode showed better quantitative performance over a wide range of specimen temperatures. These results indicate that the pulsed laser atom probe prevents both detection loss and preferential evaporation under a wide range of measurement conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Antibody tagged gold nanoparticles as scattering probes for the pico molar detection of the proteins in blood serum using nanoparticle tracking analyzer.

    Science.gov (United States)

    Kashid, Sahebrao Balaso; Tak, Rajesh D; Raut, Rajesh Warluji

    2015-09-01

    We report a rapid one-step immunoassay to detect protein using antibody conjugated gold nanoparticles (AbGNPs) where the targeted protein concentration was determined by analyzing the gold nanoparticle aggregation caused by antibody-antigen interactions using nanoparticles tracking analysis (NTA) technique. The sandwich structure constituting the binding of the targeted human IgG to the gold nanoparticle conjugates with goat anti human monoclonal IgG (AbGNPs) was confirmed by transmission electron microscopy. The binding of human IgG (antigen, mentioned hence forth as AT) induce AbGNPs to form dimers or trimers through a typical antibody-antigen-antibody sandwich structure that can be analyzed for the sensitive determination on the basis of change in hydrodynamic diameter of AbGNPs. By this method the minimum detectable concentration of AT is found to be below 2pg/ml. We expect that a significant change in the hydrodynamic diameter of AbGNP could form the basis for the rapid one-step immunoassay development. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Quantitative determination of lateral concentration and depth profile of histidine-tagged recombinant proteins probed by grazing incidence X-ray fluorescence.

    Science.gov (United States)

    Körner, Alexander; Abuillan, Wasim; Deichmann, Christina; Rossetti, Fernanda F; Köhler, Almut; Konovalov, Oleg V; Wedlich, Doris; Tanaka, Motomu

    2013-05-02

    We have demonstrated that the complementary combination of grazing incidence X-ray fluorescence (GIXF) with specular X-ray reflectivity (XRR) can be used to quantitatively determine the density profiles of Ni(2)(+) ions complexed with chelator headgroups as well as S atoms in recombinant proteins anchored to lipid monolayers at the air/water interface. First, we prepared phospholipid monolayers incorporating chelator lipid anchors at different molar fractions at the air/water interface. The fine-structures perpendicular to the global plane of monolayers were characterized by XRR in the presence of Ni(2)(+) ions, yielding the thickness, roughness, and electron density of the stratified lipid monolayers. X-ray fluorescence intensities from Ni Kα core levels recorded at the incidence angles below and above the critical angle of total reflection allow for the determination of the position and lateral density of Ni(2)(+) ions associated with chelator headgroups with a high spatial accuracy (±5 Å). The coupling of histidine-tagged Xenopus cadherin 11 (Xcad-11) can also be identified by changes in the fines-structures using XRR. Although fluorescence intensities from S Kα level were much weaker than Ni Kα signals, we could detect the location of S atoms in recombinant Xcad-11 proteins.

  16. An Investigation of Low Biofouling Copper-charged Membranes

    Science.gov (United States)

    Asapu, Sunitha

    with increased biofouling resistance. The goal of this project was to develop low-biofouling nanofiltration cellulose acetate (CA) membranes through functionalization with metal chelating ligands charged with biocidal metal ions, i.e. copper ions. To this end, glycidyl methacrylate (GMA), an epoxy, was used to attach a chelating agent, iminodiacetic acid (IDA) to facilitate the charging of copper to the membrane surface. Both CA and CA-GMA membranes were cast using the phase-inversion method. The CA-GMA membranes were then charged with copper ions to make them low biofouling. Pore size distribution analysis of CA and copper charged membranes were conducted using various molecular weights of polyethylene glycol (PEG). CA and copper-charged membranes were characterized using Fourier Transform Infrared (FTIR), contact angle to measure hydrophilicity changes, and using scanning electron microscope (SEM) coupled with X-ray energy dispersive spectroscopy EDS to monitor copper leaching. Permeation experiments were conducted with distilled (DI) water, protein solutions, and synthetic brackish water containing microorganisms. The DI water permeation of the copper-charged membranes was initially lower than the CA membranes. The membranes were then subjected to bovine serum albumin (BSA) and lipase filtration. The copper-charged membranes showed higher pure water flux values for both proteins as compared to CA membranes. The rejection of BSA and lipase was the same for both the copper charged and CA membranes. The filtration with the synthetic brackish water showed that copper-charged membranes had higher flux values as compared to CA membranes, and biofouling analysis showed more bacteria on the CA membranes as compared to copper-charged membranes. Therefore, the copper-charged membranes made here have shown a potential to be used as low-biofouling membranes in the future.

  17. Copper uptake across rainbow trout gills: mechanisms of apical entry

    DEFF Research Database (Denmark)

    Grosell, Martin Hautopp; Wood, C. M.

    2002-01-01

    Copper, Homeostasis, sodium uptake, copper/sodium interactions, gill, rainbow trout, Oncorhynchus mykiss......Copper, Homeostasis, sodium uptake, copper/sodium interactions, gill, rainbow trout, Oncorhynchus mykiss...

  18. $^{204m}$Pb: A new Probe for TDPAC Experiments in Biology Complementing the Well Established Probes $^{111}$Cd and $^{199m}$Hg

    CERN Multimedia

    2002-01-01

    The short-lived nuclear probes $\\,^{111m}$Cd( t$_{1/2}$ = 49 min) , $^{199m}$Hg ( t$_{1/2}$ = 43 min) , and $^{204m}$Pb( t$_{1/2}$ = 43 min) supplied by ISOLDE are used to study the interaction of metals with biological macromolecules like, e.g., DNA and proteins. The structure and dynamics of metal sites in biomolecules are important in determining the functional efficiency of these macromolecules. Many life processes are based on such interactions. In order to study those metal sites close to physiological conditions a highly sensitive spectroscopic method is required, like Time Differential Perturbed Angular Correlation (TDPAC). Here, a radioactive atom is placed at the site of interest and by correlating the emitted $\\gamma$-quanta in space and on a nanosecond time scale local structural information is provided via the Nuclear Quadrupole Interaction. These investigations will allow a deeper insight into the adaptivity and rigidity of metal sites in the blue copper proteins (electron transfer proteins), th...

  19. Copper induces hepatocyte injury due to the endoplasmic reticulum stress in cultured cells and patients with Wilson disease

    Energy Technology Data Exchange (ETDEWEB)

    Oe, Shinji, E-mail: ooes@med.uoeh-u.ac.jp; Miyagawa, Koichiro, E-mail: koichiro@med.uoeh-u.ac.jp; Honma, Yuichi, E-mail: y-homma@med.uoeh-u.ac.jp; Harada, Masaru, E-mail: msrharada@med.uoeh-u.ac.jp

    2016-09-10

    Copper is an essential trace element, however, excess copper is harmful to human health. Excess copper-derived oxidants contribute to the progression of Wilson disease, and oxidative stress induces accumulation of abnormal proteins. It is known that the endoplasmic reticulum (ER) plays an important role in proper protein folding, and that accumulation of misfolded proteins disturbs ER homeostasis resulting in ER stress. However, copper-induced ER homeostasis disturbance has not been fully clarified. We treated human hepatoma cell line (Huh7) and immortalized-human hepatocyte cell line (OUMS29) with copper and chemical chaperones, including 4-phenylbutyrate and ursodeoxycholic acid. We examined copper-induced oxidative stress, ER stress and apoptosis by immunofluorescence microscopy and immunoblot analyses. Furthermore, we examined the effects of copper on carcinogenesis. Excess copper induced not only oxidative stress but also ER stress. Furthermore, excess copper induced DNA damage and reduced cell proliferation. Chemical chaperones reduced this copper-induced hepatotoxicity. Excess copper induced hepatotoxicity via ER stress. We also confirmed the abnormality of ultra-structure of the ER of hepatocytes in patients with Wilson disease. These findings show that ER stress plays a pivotal role in Wilson disease, and suggests that chemical chaperones may have beneficial effects in the treatment of Wilson disease.

  20. Copper induces hepatocyte injury due to the endoplasmic reticulum stress in cultured cells and patients with Wilson disease

    International Nuclear Information System (INIS)

    Oe, Shinji; Miyagawa, Koichiro; Honma, Yuichi; Harada, Masaru

    2016-01-01

    Copper is an essential trace element, however, excess copper is harmful to human health. Excess copper-derived oxidants contribute to the progression of Wilson disease, and oxidative stress induces accumulation of abnormal proteins. It is known that the endoplasmic reticulum (ER) plays an important role in proper protein folding, and that accumulation of misfolded proteins disturbs ER homeostasis resulting in ER stress. However, copper-induced ER homeostasis disturbance has not been fully clarified. We treated human hepatoma cell line (Huh7) and immortalized-human hepatocyte cell line (OUMS29) with copper and chemical chaperones, including 4-phenylbutyrate and ursodeoxycholic acid. We examined copper-induced oxidative stress, ER stress and apoptosis by immunofluorescence microscopy and immunoblot analyses. Furthermore, we examined the effects of copper on carcinogenesis. Excess copper induced not only oxidative stress but also ER stress. Furthermore, excess copper induced DNA damage and reduced cell proliferation. Chemical chaperones reduced this copper-induced hepatotoxicity. Excess copper induced hepatotoxicity via ER stress. We also confirmed the abnormality of ultra-structure of the ER of hepatocytes in patients with Wilson disease. These findings show that ER stress plays a pivotal role in Wilson disease, and suggests that chemical chaperones may have beneficial effects in the treatment of Wilson disease.

  1. Synthesis of novel heterobimetallic copper(I) hydrazone Schiff base complexes: a comparative study on the effect of heterocyclic hydrazides towards interaction with DNA/protein, free radical scavenging and cytotoxicity.

    Science.gov (United States)

    Sathyadevi, Palanisamy; Krishnamoorthy, Paramasivam; Butorac, Rachel R; Cowley, Alan H; Dharmaraj, Nallasamy

    2012-05-01

    Two new copper(I) hydrazone complexes have been synthesised from bivalent copper precursor [CuCl(2)(PPh(3))(2)] and ferrocene containing bidentate hydrazone ligands HL(1) (1) or HL(2) (2). Based on the elemental analyses and spectroscopic data, the complexes are best formulated as [CuL(1)(PPh(3))(2)] (3) and [CuL(2)(PPh(3))(2)] (4) of the monovalent copper ion. Solid state structures of ligand 2 and its corresponding complex 4 were also determined. The DNA/albumin interactions of all the synthesised compounds were investigated using absorption, emission and synchronous fluorescence studies. Further, antioxidant properties of all the compounds have also been checked against ABTS, O(2)(-) and OH radicals. Additionally, the in vitro cytotoxic activity of compounds 1-4 was assessed using tumour (HeLa, A431) and non-tumour (NIH 3T3) cell lines.

  2. Hydrogen bond strength in membrane proteins probed by time-resolved1H-detected solid-state NMR and MD simulations.

    Science.gov (United States)

    Medeiros-Silva, João; Jekhmane, Shehrazade; Baldus, Marc; Weingarth, Markus

    2017-10-01

    1 H-detected solid-state NMR in combination with 1 H/ 2 D exchange steps allows for the direct identification of very strong hydrogen bonds in membrane proteins. On the example of the membrane-embedded potassium channel KcsA, we quantify the longevity of such very strong hydrogen bonds by combining time-resolved 1 H-detected solid-state NMR experiments and molecular dynamics simulations. In particular, we show that the carboxyl-side chain of the highly conserved residue Glu51 is involved in ultra-strong hydrogen bonds, which are fully-water-exposed and yet stable for weeks. The astonishing stability of these hydrogen bonds is important for the structural integrity of potassium channels, which we further corroborate by computational studies. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Electrical resistivity probes

    Science.gov (United States)

    Lee, Ki Ha; Becker, Alex; Faybishenko, Boris A.; Solbau, Ray D.

    2003-10-21

    A miniaturized electrical resistivity (ER) probe based on a known current-voltage (I-V) electrode structure, the Wenner array, is designed for local (point) measurement. A pair of voltage measuring electrodes are positioned between a pair of current carrying electrodes. The electrodes are typically about 1 cm long, separated by 1 cm, so the probe is only about 1 inch long. The electrodes are mounted to a rigid tube with electrical wires in the tube and a sand bag may be placed around the electrodes to protect the electrodes. The probes can be positioned in a borehole or on the surface. The electrodes make contact with the surrounding medium. In a dual mode system, individual probes of a plurality of spaced probes can be used to measure local resistance, i.e. point measurements, but the system can select different probes to make interval measurements between probes and between boreholes.

  4. The Type 3 copper site is intact but labile in Type 2-depleted laccase

    DEFF Research Database (Denmark)

    Frank, P; Farver, O; Pecht, I

    1983-01-01

    We report results of experiments designed to characterize the Type 1 and Type 3 copper sites in Rhus laccase depleted of Type 2 copper (T2D). Use of the Lowry method for determining protein concentration yielded the value 5620 +/- 570 M-1 cm-1 for the extinction of the 615-nm absorption band of t...

  5. Multilevel cycle of anthropogenic copper.

    Science.gov (United States)

    Graedel, T E; van Beers, D; Bertram, M; Fuse, K; Gordon, R B; Gritsinin, A; Kapur, A; Klee, R J; Lifset, R J; Memon, L; Rechberger, H; Spatari, S; Vexler, D

    2004-02-15

    A comprehensive contemporary cycle for stocks and flows of copper is characterized and presented, incorporating information on extraction, processing, fabrication and manufacturing, use, discard, recycling, final disposal, and dissipation. The analysis is performed on an annual basis, ca. 1994, at three discrete governmental unit levels--56 countries or country groups that together comprise essentially all global anthropogenic copper stocks and flows, nine world regions, and the planet as a whole. Cycles for all of these are presented and discussed, and a "best estimate" global copper cycle is constructed to resolve aggregation discrepancies. Among the most interesting results are (1) transformation rates and recycling rates in apparently similar national economies differ by factors of two or more (country level); (2) the discard flows that have the greatest potential for copper recycling are those with low magnitude flows but high copper concentrations--electronics, electrical equipment, and vehicles (regional level); (3) worldwide, about 53% of the copper that was discarded in various forms was recovered and reused or recycled (global level); (4) the highest rate of transfer of discarded copper to repositories is into landfills, but the annual amount of copper deposited in mine tailings is nearly as high (global level); and (5) nearly 30% of copper mining occurred merely to replace copper that was discarded. The results provide a framework for similar studies of other anthropogenic resource cycles as well as a basis for supplementary studies in resource stocks, industrial resource utilization, waste management, industrial economics, and environmental impacts.

  6. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain.

    Science.gov (United States)

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-06-24

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [(1)H, (15)N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif.

  7. and copper(II)

    Indian Academy of Sciences (India)

    Unknown

    that the imidazolate-bridged complex is stable over the pH-range 7⋅15–10⋅0. .... copper(II) complex. The observed room temperature magnetic moments are around. 1⋅79 BM, in agreement with a one-spin (S = 1/2) system. 3.2 EPR studies .... (SK) thanks the Council of Scientific & Industrial Research, New Delhi for an.

  8. Copper Pyrimidine based MOFs

    Indian Academy of Sciences (India)

    Cl..Cu.. interactions but restricted ..Cu..N N..Cu interacations. Supramolecular isomers of Br and I are reported for the first time in this paper. [Cu2I(pdz)X2]. Figure S18. Self assembly of the simultaneous presence of tecton {Cu(sol)3X} and {Cu(pdz)(sol)2X} result in [Cu2I(pdz)X2]. 1. Table S3. Copper Pyrimidine based MOFs ...

  9. Native copper as a natural analogue for copper canisters

    International Nuclear Information System (INIS)

    Marcos, N.

    1989-12-01

    This paper discusses the occurrence of native copper as found in geological formations as a stability analogue of copper canisters that are planned to be used for the disposal of spent nuclear fuel in the Finnish bedrock. A summary of several publications on native copper occurrences is presented. The present geochemical and geohydrological conditions in which copper is met with in its metallic state show that metallic copper is stable in a wide range of temperatures. At low temperatures native copper is found to be stable where groundwater has moderate pH (about 7), low Eh (< +100 mV), and low total dissolved solids, especially chloride. Microscopical and microanalytical studies were carried out on a dozen of rock samples containing native copper. The results reveal that the metal shows no significant alteration. Only the surface of copper grains is locally coated. In the oldest samples there exist small corrosion cracks; the age of the oldest samples is over 1,000 million years. A review of several Finnish groundwater studies suggests that there are places in Finland where the geohydrological conditions are favourable for native copper stability. (orig.)

  10. The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders

    Science.gov (United States)

    Fieten, Hille; Gill, Yadvinder; Martin, Alan J.; Concilli, Mafalda; Dirksen, Karen; van Steenbeek, Frank G.; Spee, Bart; van den Ingh, Ted S. G. A. M.; Martens, Ellen C. C. P.; Festa, Paola; Chesi, Giancarlo; van de Sluis, Bart; Houwen, Roderick H. J. H.; Watson, Adrian L.; Aulchenko, Yurii S.; Hodgkinson, Victoria L.; Zhu, Sha; Petris, Michael J.; Polishchuk, Roman S.; Leegwater, Peter A. J.; Rothuizen, Jan

    2016-01-01

    ABSTRACT The deleterious effects of a disrupted copper metabolism are illustrated by hereditary diseases caused by mutations in the genes coding for the copper transporters ATP7A and ATP7B. Menkes disease, involving ATP7A, is a fatal neurodegenerative disorder of copper deficiency. Mutations in ATP7B lead to Wilson disease, which is characterized by a predominantly hepatic copper accumulation. The low incidence and the phenotypic variability of human copper toxicosis hamper identification of causal genes or modifier genes involved in the disease pathogenesis. The Labrador retriever was recently characterized as a new canine model for copper toxicosis. Purebred dogs have reduced genetic variability, which facilitates identification of genes involved in complex heritable traits that might influence phenotype in both humans and dogs. We performed a genome-wide association study in 235 Labrador retrievers and identified two chromosome regions containing ATP7A and ATP7B that were associated with variation in hepatic copper levels. DNA sequence analysis identified missense mutations in each gene. The amino acid substitution ATP7B:p.Arg1453Gln was associated with copper accumulation, whereas the amino acid substitution ATP7A:p.Thr327Ile partly protected against copper accumulation. Confocal microscopy indicated that aberrant copper metabolism upon expression of the ATP7B variant occurred because of mis-localization of the protein in the endoplasmic reticulum. Dermal fibroblasts derived from ATP7A:p.Thr327Ile dogs showed copper accumulation and delayed excretion. We identified the Labrador retriever as the first natural, non-rodent model for ATP7B-associated copper toxicosis. Attenuation of copper accumulation by the ATP7A mutation sheds an interesting light on the interplay of copper transporters in body copper homeostasis and warrants a thorough investigation of ATP7A as a modifier gene in copper-metabolism disorders. The identification of two new functional variants in

  11. The Menkes and Wilson disease genes counteract in copper toxicosis in Labrador retrievers: a new canine model for copper-metabolism disorders

    Directory of Open Access Journals (Sweden)

    Hille Fieten

    2016-01-01

    Full Text Available The deleterious effects of a disrupted copper metabolism are illustrated by hereditary diseases caused by mutations in the genes coding for the copper transporters ATP7A and ATP7B. Menkes disease, involving ATP7A, is a fatal neurodegenerative disorder of copper deficiency. Mutations in ATP7B lead to Wilson disease, which is characterized by a predominantly hepatic copper accumulation. The low incidence and the phenotypic variability of human copper toxicosis hamper identification of causal genes or modifier genes involved in the disease pathogenesis. The Labrador retriever was recently characterized as a new canine model for copper toxicosis. Purebred dogs have reduced genetic variability, which facilitates identification of genes involved in complex heritable traits that might influence phenotype in both humans and dogs. We performed a genome-wide association study in 235 Labrador retrievers and identified two chromosome regions containing ATP7A and ATP7B that were associated with variation in hepatic copper levels. DNA sequence analysis identified missense mutations in each gene. The amino acid substitution ATP7B:p.Arg1453Gln was associated with copper accumulation, whereas the amino acid substitution ATP7A:p.Thr327Ile partly protected against copper accumulation. Confocal microscopy indicated that aberrant copper metabolism upon expression of the ATP7B variant occurred because of mis-localization of the protein in the endoplasmic reticulum. Dermal fibroblasts derived from ATP7A:p.Thr327Ile dogs showed copper accumulation and delayed excretion. We identified the Labrador retriever as the first natural, non-rodent model for ATP7B-associated copper toxicosis. Attenuation of copper accumulation by the ATP7A mutation sheds an interesting light on the interplay of copper transporters in body copper homeostasis and warrants a thorough investigation of ATP7A as a modifier gene in copper-metabolism disorders. The identification of two new functional

  12. Application of INEPT 109Ag and 15N NMR spectroscopy for the study of metal-ligand interaction of silver analogues of Copper(I) model compounds

    NARCIS (Netherlands)

    Koten, G. van; Stein, G.C. van; Brevard, C.

    1983-01-01

    Because of the presence of copper (in its reduced state) at active sites in protein it has become very important to study copper(I) model complexes by spectroscopic techniques.We now report that if copper(I) in model complexes can be substituted by silver(I) with retention of the structural features

  13. Excitation relaxation dynamics and energy transfer in fucoxanthin-chlorophyll a/c-protein complexes, probed by time-resolved fluorescence.

    Science.gov (United States)

    Akimoto, Seiji; Teshigahara, Ayaka; Yokono, Makio; Mimuro, Mamoru; Nagao, Ryo; Tomo, Tatsuya

    2014-09-01

    In algae, light-harvesting complexes contain specific chlorophylls (Chls) and keto-carotenoids; Chl a, Chl c, and fucoxanthin (Fx) in diatoms and brown algae; Chl a, Chl c, and peridinin in photosynthetic dinoflagellates; and Chl a, Chl b, and siphonaxanthin in green algae. The Fx-Chl a/c-protein (FCP) complex from the diatom Chaetoceros gracilis contains Chl c1, Chl c2, and the keto-carotenoid, Fx, as antenna pigments, in addition to Chl a. In the present study, we investigated energy transfer in the FCP complex associated with photosystem II (FCPII) of C. gracilis. For these investigations, we analyzed time-resolved fluorescence spectra, fluorescence rise and decay curves, and time-resolved fluorescence anisotropy data. Chl a exhibited different energy forms with fluorescence peaks ranging from 677 nm to 688 nm. Fx transferred excitation energy to lower-energy Chl a with a time constant of 300fs. Chl c transferred excitation energy to Chl a with time constants of 500-600fs (intra-complex transfer), 600-700fs (intra-complex transfer), and 4-6ps (inter-complex transfer). The latter process made a greater contribution to total Chl c-to-Chl a transfer in intact cells of C. gracilis than in the isolated FCPII complexes. The lower-energy Chl a received excitation energy from Fx and transferred the energy to higher-energy Chl a. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy. Copyright © 2014. Published by Elsevier B.V.

  14. Uptake and distribution of copper sulfate and its effect on the respiration rate of the hemocyanin-producing freshwater snail Lymnaea natalensis

    Energy Technology Data Exchange (ETDEWEB)

    Wolmarans, C.T.; Yssel, E.

    1988-08-01

    Copper sulfate was one of the earliest compounds suggested as a molluscicide and although several new compounds have since been developed, copper sulfate is still widely used against freshwater snail intermediate hosts of trematode parasites causing bilharzia. However, the toxic effect that copper sulfate may have on these species has not yet been investigated adequately. This incomplete picture of the action of copper sulfate on freshwater snails is further complicated by the fact that some of these snail species have hemocyanin (a protein containing copper) as respiration pigment. Because of the existence of a copper metabolic pathway, these species may handle external copper differently from those species with hemoglobin as respiration pigment. In the present study, the uptake of external copper in the form of copper sulfate, as well as the effect of this ion on respiration rate, was investigated in Lymnaea natalensis, the intermediate host of Fasciola gigantica. This snail possesses hemocyanin as respiratory pigment.

  15. [Incorporation of Copper Ions into T2/T3 Centers of Two-Domain Laccases].

    Science.gov (United States)

    Gabdulkhakov, A G; Kostareva, O S; Kolyadenko, I A; Mikhaylina, A O; Trubitsina, L I; Tishchenko, S V

    2018-01-01

    Laccase belongs to the family of copper-containing oxidases. A study was made of the mechanism that sustains the incorporation of copper ions into the T2/T3 centers of recombinant two-domain laccase Streptomyces griseoflavus Ac-993. The occupancy of the T3 center by copper ions was found to increase with an increasing copper content in the culture medium and after dialysis of the protein preparation against a copper sulfate-containing buffer. The T2 center was filled only when overproducer strain cells were grown at a higher copper concentration in the medium. Two-domain laccases were assumed to possess a channel that serves to deliver copper ions to the T3 center during the formation of the three-dimensional laccase conformation and dialysis of the protein preparation. A narrower channel leads to the T2 center in two-domain laccases compared with three-domain ones, rendering the center less accessible for copper atoms. The incorporation of copper ions into the T2 center of two-domain laccases is likely to occur in the course of their biosynthesis or the formation of a functional trimer.

  16. Neurokinin B and serum albumin limit copper binding to mammalian gonadotropin releasing hormone.

    Science.gov (United States)

    Gul, Ahmad Samir; Tran, Kevin K; Jones, Christopher E

    2018-02-26

    Gonadotropin releasing hormone (GnRH) triggers secretion of luteinizing hormone and follicle stimulating hormone from gonadotropic cells in the anterior pituitary gland. GnRH is able to bind copper, and both in vitro and in vivo studies have suggested that the copper-GnRH complex is more potent at triggering gonadotropin release than GnRH alone. However, it remains unclear whether copper-GnRH is the active species in vivo. To explore this we have estimated the GnRH-copper affinity and have examined whether GnRH remains copper-bound in the presence of serum albumin and the neuropeptide neurokinin B, both copper-binding proteins that GnRH will encounter in vivo. We show that GnRH has a copper dissociation constant of ∼0.9 × 10 -9  M, however serum albumin and neurokinin B can extract metal from the copper-GnRH complex. It is therefore unlikely that a copper-GnRH complex will survive transit through the pituitary portal circulation and that any effect of copper must occur outside the bloodstream in the absence of neurokinin B. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Copper deprivation modulates CTR1 and CUP1 expression and enhances cisplatin cytotoxicity in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kommuguri, Upendra Nadh; Bodiga, Sreedhar; Sankuru, Suneetha; Bodiga, Vijaya Lakshmi

    2012-01-01

    Saccharomyces cerevisiae has been established as a model system for cancer studies, due to the widely conserved family of genes involved in cell cycle progression, proliferation and apoptosis. In the current study, we sought to determine whether copper deprivation modulates sensitivity of yeast to cisplatin. Yeast cultures grown in low copper medium and exposed to bathocuproiene disulfate (BCS) resulted in significant reduction of intracellular copper. We report here that low copper medium rendered BY4741 hypersensitive to cisplatin (CDDP). Yeast grown in low copper medium exhibited ∼2.0 fold enhanced cytotoxicity in survival and colony-forming ability, compared to copper adequate control cells grown in YPD. The effect of copper restriction on CDDP sensitivity appeared to be associated with the up regulation of CTR1, facilitating enhanced uptake and accumulation of CDDP. Also, CDDP further lowered copper deprivation-induced changes in CUP1 metallothionein levels, SOD activity and GSH levels. These changes were associated with increased protein oxidation and lipid peroxidation induced by CDDP. These results thus suggest that cisplatin cytotoxicity is potentiated under low copper conditions due to enhanced uptake and accumulation of cisplatin and also in part due to lowered antioxidant defense and increased oxidative stress imposed by copper deprivation. Copyright © 2011 Elsevier GmbH. All rights reserved.

  18. 5 prime -Azido-(3,6- sup 3 H sub 2 )-1-naphthylphthalamic acid, a photoactivatable probe for naphthylphthalamic acid receptor proteins from higher plants: Identification of a 23-kDa protein from maize coleoptile plasma membranes

    Energy Technology Data Exchange (ETDEWEB)

    Zettl, R.; Feldwisch, J.; Schell, J.; Palme, K. (Max-Planck-Inst. fuer Zuechtungsforschung, Koeln (West Germany)); Boland, W. (Univ. Karlsruhe (West Germany))

    1992-01-15

    1-Naphthylphthalamic acid (NPA) is a specific inhibitor of polar auxin transport that blocks carrier mediated auxin efflux from plant cells. To allow identification of the NPA receptor thought to be part of the auxin efflux carrier, the authors have synthesized a tritiated, photolabile NPA analogue, 5{prime}-azido-(3,6-{sup 3}H{sub 2})NPA (({sup 3}H{sub 2})N{sub 3}NPA). This analogue was used to identify NPA-binding proteins in fractions highly enriched for plasma membrane vesicles isolated from maize coleoptiles (Zea mays L.). Competition studies showed that binding of ({sup 3}H{sub 2})N{sub 3}NPA to maize plasma membrane vesicles was blocked by nonradioactive NPA but not by benzoic acid. After incubation of plasma membrane vesicles with ({sup 3}H{sub 2})N{sub 3}NPA and exposure to UV light, they observed specific photoaffinity labeling of a protein with an apparent molecular mass of 23 kDa. Pretreatment of the plasma membrane vesicles with indole-3-acetic acid or with the auxin-transport inhibitors NPA and 2,3,5-triiodobenzoic acid strongly reduced specific labeling of this protein. This 23-kDa protein was also labeled by addition of 5-azido-(7-{sup 3}H)indole-3-acetic acid to plasma membranes prior to exposure to UV light. The 23-kDa protein was solubilized from plasma membranes by 1% Triton X-100. The possibility that this 23-kDa polypeptide is part of the auxin efflux carrier system is discussed.

  19. High temperature probe

    Science.gov (United States)

    Swan, Raymond A.

    1994-01-01

    A high temperature probe for sampling, for example, smokestack fumes, and is able to withstand temperatures of 3000.degree. F. The probe is constructed so as to prevent leakage via the seal by placing the seal inside the water jacket whereby the seal is not exposed to high temperature, which destroys the seal. The sample inlet of the probe is also provided with cooling fins about the area of the seal to provide additional cooling to prevent the seal from being destroyed. Also, a heated jacket is provided for maintaining the temperature of the gas being tested as it passes through the probe. The probe includes pressure sensing means for determining the flow velocity of an efficient being sampled. In addition, thermocouples are located in various places on the probe to monitor the temperature of the gas passing there through.

  20. Microneurosurgical water probe.

    Science.gov (United States)

    Pogády, P; Wurm, G

    2005-04-01

    When constructing the micro-neurosurgical water ball probe, the authors have simply combined the properties of a ball probe with an irrigational function and the supportive role of water current to form a new irrigating ball dissector. The micro-instrument has an outlet mechanism with which the surgeon can regulate the flow of physiological solution into the operational field. Its point has the properties of a ball probe, and the overall bayonet shape facilitates surgical interventions in deep tissues under microscopic control. The water probe therefore enables the surgeon to perform precise mechanical preparation supported by a regulated current of water and a targeted irrigation in the operational field. The physiological solution in the pressure infusion cuff is under minimal pressure and directly connected to the probe. Due to the fact that one device can be used for various purposes the water ball probe represents an advantageous alternative to conventional micro-neurosurgical preparation.

  1. Bioaccumulation of copper ions by Escherichia coli expressing vanabin genes from the vanadium-rich ascidian Ascidia sydneiensis samea.

    Science.gov (United States)

    Ueki, Tatsuya; Sakamoto, Yasuhisa; Yamaguchi, Nobuo; Michibata, Hitoshi

    2003-11-01

    The genes encoding two vanadium-binding proteins, vanabin1 and vanabin2, from a vanadium-rich ascidian, Ascidia sydneiensis samea, were recently identified and cloned (T. Ueki, T. Adachi, S. Kawano, M. Aoshima, N. Yamaguchi, K. Kanamori, and H. Michibata, Biochim. Biophys. Acta 1626:43-50, 2003). The vanabins were found to bind vanadium(IV), and an excess of copper(II) ions inhibited the binding of vanadium(IV) to the vanabins in vitro. In this study, we constructed Escherichia coli strains that expressed vanabin1 or vanabin2 fused to maltose-binding protein (MBP) in the periplasmic space. We found that both strains accumulated about twenty times more copper(II) ions than the control BL21 strain, while no significant accumulation of vanadium was observed. The strains expressing either MBP-vanabin1 or MBP-vanabin2 absorbed approximately 70% of the copper ions in the medium to which 10 micro M copper (II) ions were initially added. The MBP-vanabin1 and MBP-vanabin2 protein expressed in the periplasm bound to copper ions at a copper:protein molar ratio of 8:1 and 5:1, respectively, but MBP did not bind to copper ions. These data showed that the metal-binding proteins vanabin1 and vanabin2 bound copper ions directly and enhanced the bioaccumulation of copper ions by E. coli.

  2. Tissue Distribution of Kir7.1 Inwardly Rectifying K+ Channel Probed in a Knock-in Mouse Expressing a Haemagglutinin-Tagged Protein

    Directory of Open Access Journals (Sweden)

    Isabel Cornejo

    2018-04-01

    Full Text Available Kir7.1 encoded by the Kcnj13 gene in the mouse is an inwardly rectifying K+ channel present in epithelia where it shares membrane localization with the Na+/K+-pump. Further investigations of the localisation and function of Kir7.1 would benefit from the availability of a knockout mouse, but perinatal mortality attributed to cleft palate in the neonate has thwarted this research. To facilitate localisation studies we now use CRISPR/Cas9 technology to generate a knock-in mouse, the Kir7.1-HA that expresses the channel tagged with a haemagglutinin (HA epitope. The availability of antibodies for the HA epitope allows for application of western blot and immunolocalisation methods using widely available anti-HA antibodies with WT tissues providing unambiguous negative control. We demonstrate that Kir7.1-HA cloned from the choroid plexus of the knock-in mouse has the electrophysiological properties of the native channel, including characteristically large Rb+ currents. These large Kir7.1-mediated currents are accompanied by abundant apical membrane Kir7.1-HA immunoreactivity. WT-controlled western blots demonstrate the presence of Kir7.1-HA in the eye and the choroid plexus, trachea and lung, and intestinal epithelium but exclusively in the ileum. In the kidney, and at variance with previous reports in the rat and guinea-pig, Kir7.1-HA is expressed in the inner medulla but not in the cortex or outer medulla. In isolated tubules immunoreactivity was associated with inner medulla collecting ducts but not thin limbs of the loop of Henle. Kir7.1-HA shows basolateral expression in the respiratory tract epithelium from trachea to bronchioli. The channel also appears basolateral in the epithelium of the nasal cavity and nasopharynx in newborn animals. We show that HA-tagged Kir7.1 channel introduced in the mouse by a knock-in procedure has functional properties similar to the native protein and the animal thus generated has clear advantages in localisation

  3. Engineering of blood vessel patterns by angio-morphogens [angiotropins]: non-mitogenic copper-ribonucleoprotein cytokins [CuRNP ribokines] with their metalloregulated constituents of RAGE-binding S100-EF-hand proteins and extracellular RNA bioaptamers in vascular remodeling of tissue and angiogenesis in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wissler, J.H. [ARCONS Applied Research, Bad Nauheim (Germany)

    2001-12-01

    Tissue vascularization is requisite to successful cell-based therapies, biomaterial design and implant integration. Thus, known problems in ossointegration of avascular implants in connection with the generation of bone tissue reflect arrays of general problems of socio-economic relevance existing in reparative medicine still waiting for to be solved. For this purpose, morphogenesis and remodeling of endothelial angio-architectures in tissue and in vitro by isolated non-mitogenic angio-morphogens [angiotropins] are considered in terms of their structure, function and action mechanisms. Extracellular angiotropins are secreted by activated leukocytes/monocytes/macrophages. They are a family of cytokines with morphogen bioactivity selectively directed to endothelial cells. Their structure was deciphered as metalloregulated copper-ribonucleoproteins [CuRNP ribokines]. They are built up of angiotropin-related S100-EF-hand protein [ARP] and highly modified and edited 5'end-phosphorylated RNA [ARNA], complexed together by copper ions. Oxidant-sensitive ARNA and their precursors represent novel types in a RNA world: They are the first isolated and sequenced forms of extracellular RNA [eRNA], may act as cytokine and bioaptamer, contain isoguanosine [crotonoside] as modified nucleoside and show up copper as RNA-structuring transition metal ion. By metalloregulated bioaptamer functions, ARNA impart novel biofunctions to RAGE-binding S100-EF-hand proteins. Angiotropin morphogens were shown suitable for neointiation and remodeling of blood vessel patterns in different, adult, embryonal and artificial tissues. These neovascular patterns manifest regulated hemodynamics for preventing tissue necrosis, supporting tissue functions and promoting wound healing. As evaluated in skin and muscle vascularization, the neovascular patterns are integrated into homeostatic control mechanisms of tissue. Thus, the morphogens show up beneficial perspectives and are suggested useful tools

  4. Recombinant foot-and-mouth disease virus (FMDV) non-structural protein 3A fused to enhanced green fluorescent protein (EGFP) as a candidate probe to identify FMDV-infected cattle in serosurveys.

    Science.gov (United States)

    Lotufo, Cecilia M; Bergmann, Ingrid E; Mattion, Nora M; Wilda, Maximiliano; Grigera, Pablo R

    2017-08-01

    Recombinant protein 3A-EGFP, a fusion construct between foot-and-mouth disease virus (FMDV) non-structural protein 3A and the enhanced green fluorescent protein (EGFP) was expressed in BL21-DE3 cells. The identity of the partially purified protein 3A-EGFP was confirmed by its reactivity with sera from cattle infected with FMDV and with a monoclonal antibody specific for FMDV-3ABC (MAb3H7) in Western blot assays. No reactivity was observed with sera from uninfected vaccinated animals. The performance of 3A-EGFP as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) was assessed and compared with that of a previously developed and validated capture ELISA that uses a 3ABC recombinant antigen (3ABC ELISA) and has been widely applied for serological surveys in Argentina. Parallel analysis of strongly and weakly positive reference sera from infected animals and 329 serum samples from uninfected vaccinated cattle showed that the 3A-EGFP antigen unequivocally identifies sera from FMDV-infected cattle with similar performance to its 3ABC counterpart. The 3A-EGFP ELISA is simpler and faster to perform than the 3ABC ELISA, since it does not require a capture step with a specific antibody. Moreover, the expression and storage of the recombinant 3A-EGFP is simplified by the absence of residual autoproteolytic activity associated to the 3C sequence. We conclude that the 3A-EGFP ELISA constitutes a promising screening method in serosurveys to determine whether or not animals are infected with FMDV.

  5. Single and Combined Exposure to Zinc- and Copper-Containing Welding Fumes Lead to Asymptomatic Systemic Inflammation.

    Science.gov (United States)

    Markert, Agnieszka; Baumann, Ralf; Gerhards, Benjamin; Gube, Monika; Kossack, Veronika; Kraus, Thomas; Brand, Peter

    2016-02-01

    Recently, it has been shown that exposure to welding fumes containing both zinc and copper leads to asymptomatic systemic inflammation in humans as shown by an increase of blood C-reactive protein. In the present study, it was investigated which metal is responsible for this effect. Fifteen healthy male subjects were exposed under controlled conditions to welding fumes containing either zinc, or copper, or copper and zinc. For each exposure blood C-reactive protein increased. Copper- and zinc-containing welding fumes are able to induce systemic inflammation.

  6. Detention of copper by sulfur nanoparticles inhibits the proliferation of A375 malignant melanoma and MCF-7 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Hao [Department of Chemistry, Jinan University, Guangzhou (China); Zhang, Yikai [Institute of Hematology, Jinan University, Guangzhou (China); Zheng, Shanyuan [School of Life Sciences, The Chinese University of Hong Kong, Hong Kong (China); Weng, Zeping; Ma, Jun [First Affiliated Hospital, Jinan University, Guangzhou (China); Li, Yangqiu [Institute of Hematology, Jinan University, Guangzhou (China); First Affiliated Hospital, Jinan University, Guangzhou (China); Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, 510632 (China); Xie, Xinyuan [Department of Chemistry, Jinan University, Guangzhou (China); Zheng, Wenjie, E-mail: tzhwj@jnu.edu.cn [Department of Chemistry, Jinan University, Guangzhou (China)

    2016-09-02

    Selective induction of cell death or growth inhibition of cancer cells is the future of chemotherapy. Clinical trials have found that cancer tissues are enriched with copper. Based on this finding, many copper-containing compounds and complexes have been designed to “copper” cancer cells using copper as bait. However, recent studies have demonstrated that copper boosts tumor development, and copper deprivation from serum was shown to effectively inhibit the promotion of cancer. Mechanistically, copper is an essential cofactor for mitogen-activated protein kinase (MAPK)/extracellular activating kinase (ERK) kinase (MEK), a central molecule in the BRAF/MEK/ERK pathway. Therefore, depleting copper from cancer cells by directly sequestering copper has a wider field for research and potential for combination therapy. Based on the affinity between sulfur and copper, we therefore designed sulfur nanoparticles (Nano-S) that detain copper, achieving tumor growth restriction. We found that spherical Nano-S could effectively bind copper and form a tighter surficial structure. Moreover, this Nano-S detention of copper effectively inhibited the proliferation of A375 melanoma and MCF-7 breast cancer cells with minimum toxicity to normal cells. Mechanistic studies revealed that Nano-S triggered inactivation of the MEK-ERK pathway followed by inhibition of the proliferation of the A375 and MCF-7 cells. In addition, lower Nano-S concentrations and shorter exposure stimulated the expression of a copper transporter as compensation, which further increased the cellular uptake and anticancer activities of cisplatin. Collectively, our results highlight the potential of Nano-S as an anticancer agent or adjuvant through its detention of copper. - Highlights: • Nano-S selectively inhibited the mitosis of A375 and MCF-7 cells by depleting copper. • Nano-S inactivated MEK/ERK pathway through the detention of copper. • Nano-S improved the cellular uptake and anticancer activities

  7. Detention of copper by sulfur nanoparticles inhibits the proliferation of A375 malignant melanoma and MCF-7 breast cancer cells

    International Nuclear Information System (INIS)

    Liu, Hao; Zhang, Yikai; Zheng, Shanyuan; Weng, Zeping; Ma, Jun; Li, Yangqiu; Xie, Xinyuan; Zheng, Wenjie

    2016-01-01

    Selective induction of cell death or growth inhibition of cancer cells is the future of chemotherapy. Clinical trials have found that cancer tissues are enriched with copper. Based on this finding, many copper-containing compounds and complexes have been designed to “copper” cancer cells using copper as bait. However, recent studies have demonstrated that copper boosts tumor development, and copper deprivation from serum was shown to effectively inhibit the promotion of cancer. Mechanistically, copper is an essential cofactor for mitogen-activated protein kinase (MAPK)/extracellular activating kinase (ERK) kinase (MEK), a central molecule in the BRAF/MEK/ERK pathway. Therefore, depleting copper from cancer cells by directly sequestering copper has a wider field for research and potential for combination therapy. Based on the affinity between sulfur and copper, we therefore designed sulfur nanoparticles (Nano-S) that detain copper, achieving tumor growth restriction. We found that spherical Nano-S could effectively bind copper and form a tighter surficial structure. Moreover, this Nano-S detention of copper effectively inhibited the proliferation of A375 melanoma and MCF-7 breast cancer cells with minimum toxicity to normal cells. Mechanistic studies revealed that Nano-S triggered inactivation of the MEK-ERK pathway followed by inhibition of the proliferation of the A375 and MCF-7 cells. In addition, lower Nano-S concentrations and shorter exposure stimulated the expression of a copper transporter as compensation, which further increased the cellular uptake and anticancer activities of cisplatin. Collectively, our results highlight the potential of Nano-S as an anticancer agent or adjuvant through its detention of copper. - Highlights: • Nano-S selectively inhibited the mitosis of A375 and MCF-7 cells by depleting copper. • Nano-S inactivated MEK/ERK pathway through the detention of copper. • Nano-S improved the cellular uptake and anticancer activities

  8. Anticancer activity of Ficus religiosa engineered copper oxide nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Sankar, Renu; Maheswari, Ramasamy; Karthik, Selvaraju [Department of Biochemistry, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamilnadu (India); Shivashangari, Kanchi Subramanian, E-mail: shivashangari@gmail.com [Regional Forensic Science Laboratory, Tiruchirapalli, Tamilnadu (India); Ravikumar, Vilwanathan, E-mail: ravikumarbdu@gmail.com [Department of Biochemistry, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamilnadu (India)

    2014-11-01

    The design, synthesis, characterization and application of biologically synthesized nanomaterials have become a vital branch of nanotechnology. There is a budding need to develop a method for environmentally benign metal nanoparticle synthesis, that do not use toxic chemicals in the synthesis protocols to avoid adverse effects in medical applications. Here, it is a report on an eco-friendly process for rapid synthesis of copper oxide nanoparticles using Ficus religiosa leaf extract as reducing and protecting agent. The synthesized copper oxide nanoparticles were confirmed by UV–vis spectrophotometer, absorbance peaks at 285 nm. The copper oxide nanoparticles were analyzed with field emission-scanning electron microscope (FE-SEM), Fourier transform infrared (FT-IR) spectroscopy, dynamic light scattering (DLS) and X-ray diffraction (XRD) spectrum. The FE-SEM and DLS analyses exposed that copper oxide nanoparticles are spherical in shape with an average particle size of 577 nm. FT-IR spectral analysis elucidates the occurrence of biomolecules required for the reduction of copper oxide ions. Zeta potential studies showed that the surface charge of the formed nanoparticles was highly negative. The XRD pattern revealed that synthesized nanoparticles are crystalline in nature. Further, biological activities of the synthesized nanoparticles were confirmed based on its stable anti-cancer effects. The apoptotic effect of copper oxide nanoparticles is mediated by the generation of reactive oxygen species (ROS) involving the disruption of mitochondrial membrane potential (Δψm) in A549 cells. The observed characteristics and results obtained in our in vitro assays suggest that the copper nanoparticles might be a potential anticancer agent. - Highlights: • Biogenic synthesis of copper oxide nanoparticles by leaf extract of Ficus religiosa • Characterized via UV–vis, FT-IR, DLS, FE-SEM with EDAX and XRD • Protein may act as an encapsulating, reducing and stabilizing

  9. Anticancer activity of Ficus religiosa engineered copper oxide nanoparticles

    International Nuclear Information System (INIS)

    Sankar, Renu; Maheswari, Ramasamy; Karthik, Selvaraju; Shivashangari, Kanchi Subramanian; Ravikumar, Vilwanathan

    2014-01-01

    The design, synthesis, characterization and application of biologically synthesized nanomaterials have become a vital branch of nanotechnology. There is a budding need to develop a method for environmentally benign metal nanoparticle synthesis, that do not use toxic chemicals in the synthesis protocols to avoid adverse effects in medical applications. Here, it is a report on an eco-friendly process for rapid synthesis of copper oxide nanoparticles using Ficus religiosa leaf extract as reducing and protecting agent. The synthesized copper oxide nanoparticles were confirmed by UV–vis spectrophotometer, absorbance peaks at 285 nm. The copper oxide nanoparticles were analyzed with field emission-scanning electron microscope (FE-SEM), Fourier transform infrared (FT-IR) spectroscopy, dynamic light scattering (DLS) and X-ray diffraction (XRD) spectrum. The FE-SEM and DLS analyses exposed that copper oxide nanoparticles are spherical in shape with an average particle size of 577 nm. FT-IR spectral analysis elucidates the occurrence of biomolecules required for the reduction of copper oxide ions. Zeta potential studies showed that the surface charge of the formed nanoparticles was highly negative. The XRD pattern revealed that synthesized nanoparticles are crystalline in nature. Further, biological activities of the synthesized nanoparticles were confirmed based on its stable anti-cancer effects. The apoptotic effect of copper oxide nanoparticles is mediated by the generation of reactive oxygen species (ROS) involving the disruption of mitochondrial membrane potential (Δψm) in A549 cells. The observed characteristics and results obtained in our in vitro assays suggest that the copper nanoparticles might be a potential anticancer agent. - Highlights: • Biogenic synthesis of copper oxide nanoparticles by leaf extract of Ficus religiosa • Characterized via UV–vis, FT-IR, DLS, FE-SEM with EDAX and XRD • Protein may act as an encapsulating, reducing and stabilizing

  10. Activation of dioxygen by copper metalloproteins and insights from model complexes.

    Science.gov (United States)

    Quist, David A; Diaz, Daniel E; Liu, Jeffrey J; Karlin, Kenneth D

    2017-04-01

    Nature uses dioxygen as a key oxidant in the transformation of biomolecules. Among the enzymes that are utilized for these reactions are copper-containing metalloenzymes, which are responsible for important biological functions such as the regulation of neurotransmitters, dioxygen transport, and cellular respiration. Enzymatic and model system studies work in tandem in order to gain an understanding of the fundamental reductive activation of dioxygen by copper complexes. This review covers the most recent advancements in the structures, spectroscopy, and reaction mechanisms for dioxygen-activating copper proteins and relevant synthetic models thereof. An emphasis has also been placed on cofactor biogenesis, a fundamentally important process whereby biomolecules are post-translationally modified by the pro-enzyme active site to generate cofactors which are essential for the catalytic enzymatic reaction. Significant questions remaining in copper-ion-mediated O 2 -activation in copper proteins are addressed.

  11. Nickel, copper and cobalt coalescence in copper cliff converter slag

    Directory of Open Access Journals (Sweden)

    Wolf A.

    2016-01-01

    Full Text Available The aim of this investigation is to assess the effect of various additives on coalescence of nickel, copper and cobalt from slags generated during nickel extraction. The analyzed fluxes were silica and lime while examined reductants were pig iron, ferrosilicon and copper-silicon compound. Slag was settled at the different holding temperatures for various times in conditions that simulated the industrial environment. The newly formed matte and slag were characterized by their chemical composition and morphology. Silica flux generated higher partition coefficients for nickel and copper than the addition of lime. Additives used as reducing agents had higher valuable metal recovery rates and corresponding partition coefficients than fluxes. Microstructural studies showed that slag formed after adding reductants consisted of primarily fayalite, with some minute traces of magnetite as the secondary phase. Addition of 5 wt% of pig iron, ferrosilicon and copper-silicon alloys favored the formation of a metallized matte which increased Cu, Ni and Co recoveries. Addition of copper-silicon alloys with low silicon content was efficient in copper recovery but coalescence of the other metals was low. Slag treated with the ferrosilicon facilitated the highest cobalt recovery while copper-silicon alloys with silicon content above 10 wt% resulted in high coalescence of nickel and copper, 87 % and 72 % respectively.

  12. Spectrographic determination of impurities in copper and copper oxide

    International Nuclear Information System (INIS)

    Sabato, S.F.; Lordello, A.R.

    1990-11-01

    An emission spectrographic method for the determination of Al, Bi, Ca, Cd, Cr, Fe, Ge, Mg, Mn, Mo, Ni, Pb, Sb, Si, Sn and Zn in copper and copper oxide is described. Two mixtures (Graphite and ZnO: graphite and GeO sub(2)) were used as buffers. The standard deviation lies around 10%. (author)

  13. Synergistic Effects of Copper Sites on Apparent Stability of Multicopper Oxidase, Fet3p

    Directory of Open Access Journals (Sweden)

    Erik Sedlák

    2018-01-01

    Full Text Available Saccharomyces cerevisiae Fet3p is a multicopper oxidase that contains three cupredoxin-like domains and four copper ions located in three distinct metal sites (T1 in domain 3; T2 and the binuclear T3 at the interface between domains 1 and 3. To probe the role of the copper sites in Fet3p thermodynamic stability, we performed urea-induced unfolding experiments with holo-, apo- and three partially-metallated (T1, T2 and T1/T2 sites depleted of copper forms of Fet3p. Using a combination of spectroscopic probes (circular dichroism, fluorescence intensity and maximum, 8-anilinonaphthalene-1-sulfonic acid (ANS emission, oxidase activity and blue color, we reveal that all forms of Fet3p unfold in a four-state reaction with two partially-folded intermediates. Using phase diagrams, it emerged that Fet3p with all copper sites filled had a significantly higher stability as compared to the combined contributions of the individual copper sites. Hence, there is long-range inter-domain communication between distal copper sites that contribute to overall Fet3p stability.

  14. Inactivation of norovirus on dry copper alloy surfaces.

    Directory of Open Access Journals (Sweden)

    Sarah L Warnes

    Full Text Available Noroviruses (family Caliciviridae are the primary cause of viral gastroenteritis worldwide. The virus is highly infectious and touching contaminated surfaces can contribute to infection spread. Although the virus was identified over 40 years ago the lack of methods to assess infectivity has hampered the study of the human pathogen. Recently the murine virus, MNV-1, has successfully been used as a close surrogate. Copper alloys have previously been shown to be effective antimicrobial surfaces against a range of bacteria and fungi. We now report rapid inactivation of murine norovirus on alloys, containing over 60% copper, at room temperature but no reduction of infectivity on stainless steel dry surfaces in simulated wet fomite and dry touch contamination. The rate of inactivation was initially very rapid and proportional to copper content of alloy tested. Viral inactivation was not as rapid on brass as previously observed for bacteria but copper-nickel alloy was very effective. The use of chelators and quenchers of reactive oxygen species (ROS determined that Cu(II and especially Cu(I ions are still the primary effectors of toxicity but quenching superoxide and hydroxyl radicals did not confer protection. This suggests Fenton generation of ROS is not important for the inactivation mechanism. One of the targets of copper toxicity was the viral genome and a reduced copy number of the gene for a viral encoded protein, VPg (viral-protein-genome-linked, which is essential for infectivity, was observed following contact with copper and brass dry surfaces. The use of antimicrobial surfaces containing copper in high risk closed environments such as cruise ships and care facilities could help to reduce the spread of this highly infectious and costly pathogen.

  15. Inactivation of Norovirus on Dry Copper Alloy Surfaces

    Science.gov (United States)

    Warnes, Sarah L.; Keevil, C. William

    2013-01-01

    Noroviruses (family Caliciviridae) are the primary cause of viral gastroenteritis worldwide. The virus is highly infectious and touching contaminated surfaces can contribute to infection spread. Although the virus was identified over 40 years ago the lack of methods to assess infectivity has hampered the study of the human pathogen. Recently the murine virus, MNV-1, has successfully been used as a close surrogate. Copper alloys have previously been shown to be effective antimicrobial surfaces against a range of bacteria and fungi. We now report rapid inactivation of murine norovirus on alloys, containing over 60% copper, at room temperature but no reduction of infectivity on stainless steel dry surfaces in simulated wet fomite and dry touch contamination. The rate of inactivation was initially very rapid and proportional to copper content of alloy tested. Viral inactivation was not as rapid on brass as previously observed for bacteria but copper-nickel alloy was very effective. The use of chelators and quenchers of reactive oxygen species (ROS) determined that Cu(II) and especially Cu(I) ions are still the primary effectors of toxicity but quenching superoxide and hydroxyl radicals did not confer protection. This suggests Fenton generation of ROS is not important for the inactivation mechanism. One of the targets of copper toxicity was the viral genome and a reduced copy number of the gene for a viral encoded protein, VPg (viral-protein-genome-linked), which is essential for infectivity, was observed following contact with copper and brass dry surfaces. The use of antimicrobial surfaces containing copper in high risk closed environments such as cruise ships and care facilities could help to reduce the spread of this highly infectious and costly pathogen. PMID:24040380

  16. Sensitive detection of mercury and copper ions by fluorescent DNA/Ag nanoclusters in guanine-rich DNA hybridization.

    Science.gov (United States)

    Peng, Jun; Ling, Jian; Zhang, Xiu-Qing; Bai, Hui-Ping; Zheng, Liyan; Cao, Qiu-E; Ding, Zhong-Tao

    2015-02-25

    In this work, we designed a new fluorescent oligonucleotides-stabilized silver nanoclusters (DNA/AgNCs) probe for sensitive detection of mercury and copper ions. This probe contains two tailored DNA sequence. One is a signal probe contains a cytosine-rich sequence template for AgNCs synthesis and link sequence at both ends. The other is a guanine-rich sequence for signal enhancement and link sequence complementary to the link sequence of the signal probe. After hybridization, the fluorescence of hybridized double-strand DNA/AgNCs is 200-fold enhanced based on the fluorescence enhancement effect of DNA/AgNCs in proximity of guanine-rich DNA sequence. The double-strand DNA/AgNCs probe is brighter and stable than that of single-strand DNA/AgNCs, and more importantly, can be used as novel fluorescent probes for detecting mercury and copper ions. Mercury and copper ions in the range of 6.0-160.0 and 6-240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. Our results indicated that the analytical parameters of the method for mercury and copper ions detection are much better than which using a single-strand DNA/AgNCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Effects of copper stress on antioxidative enzymes, chlorophyll and ...

    African Journals Online (AJOL)

    Administrator

    2011-09-05

    Sep 5, 2011 ... Our study showed the effect of Cu on Atriplex halimus grown in hydroponics conditions. The aim of this work was to investigate some enzymatic systems response of this plant to copper stress. Analysis was carried on enzymatic profiles, protein tenor and chlorophyll content of A. halimus leaves. Two.

  18. Effects of copper stress on antioxidative enzymes, chlorophyll and ...

    African Journals Online (AJOL)

    Our study showed the effect of Cu on Atriplex halimus grown in hydroponics conditions. The aim of this work was to investigate some enzymatic systems response of this plant to copper stress. Analysis was carried on enzymatic profiles, protein tenor and chlorophyll content of A. halimus leaves. Two months after sowing, ...

  19. Antimicrobial electrospun silver-, copper- and zinc-doped polyvinylpyrrolidone nanofibers

    Energy Technology Data Exchange (ETDEWEB)

    Quirós, Jennifer [Department of Chemical Engineering, University of Alcalá, 28871 Alcalá de Henares, Madrid (Spain); Borges, João P. [CENIMAT/I3N, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Boltes, Karina [Department of Chemical Engineering, University of Alcalá, 28871 Alcalá de Henares, Madrid (Spain); Madrid Institute for Advanced Studies of Water (IMDEA Agua), Parque Científico Tecnológico, E-28805, Alcalá de Henares, Madrid (Spain); Rodea-Palomares, Ismael [Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid (Spain); Rosal, Roberto [Department of Chemical Engineering, University of Alcalá, 28871 Alcalá de Henares, Madrid (Spain); Madrid Institute for Advanced Studies of Water (IMDEA Agua), Parque Científico Tecnológico, E-28805, Alcalá de Henares, Madrid (Spain)

    2015-12-15

    Highlights: • Electrospun polyvinylpyrrolidone (PVP) nanofibers containing silver, copper, and zinc. • Antimicrobial effect for the bacteria Staphylococcus aureus and Escherichia coli. • Silver strongly reduced colony forming units and bacterial viability. • Silver, copper, and zinc led to a significant increase of non-viable cells on mats. - Abstract: The use of electrospun polyvinylpyrrolidone (PVP) nanofibers containing silver, copper, and zinc nanoparticles was studied to prepare antimicrobial mats using silver and copper nitrates and zinc acetate as precursors. Silver became reduced during electrospinning and formed nanoparticles of several tens of nanometers. Silver nanoparticles and the insoluble forms of copper and zinc were dispersed using low molecular weight PVP as capping agent. High molecular weight PVP formed uniform fibers with a narrow distribution of diameters around 500 nm. The fibers were converted into an insoluble network using ultraviolet irradiation crosslinking. The efficiency of metal-loaded mats against the bacteria Escherichia coli and Staphylococcus aureus was tested for different metal loadings by measuring the inhibition of colony forming units and the staining with fluorescent probes for metabolic viability and compromised membranes. The assays included the culture in contact with mats and the direct staining of surface attached microorganisms. The results indicated a strong inhibition for silver-loaded fibers and the absence of significant amounts of viable but non-culturable microorganisms. Copper and zinc-loaded mats also decreased the metabolic activity and cell viability, although in a lesser extent. Metal-loaded fibers allowed the slow release of the soluble forms of the three metals.

  20. Probe tests microweld strength

    Science.gov (United States)

    1965-01-01

    Probe is developed to test strength of soldered, brazed or microwelded joints. It consists of a spring which may be adjusted to the desired test pressure by means of a threaded probe head, and an indicator lamp. Device may be used for electronic equipment testing.

  1. Two-step flash light sintering process for enhanced adhesion between copper complex ion/silane ink and a flexible substrate

    International Nuclear Information System (INIS)

    Jeon, Eun-Beom; Joo, Sung-Jun; Ahn, Heejoon; Kim, Hak-Sung

    2016-01-01

    A copper complex ion ink (including copper nanoparticles, a copper precursor and a silane coupling agent) was synthesized to enhance the adhesion between the copper pattern and a polyimide (PI) substrate. Oxygen plasma treatment was performed on the polyimide substrate to initiate a chemical reaction between the complex ion ink and the polyimide. Then, a two-step flash light sintering method (consisting of preheating and main sintering) was used to sinter the copper complex ion ink. The copper complex ion patterns were characterized as a function of the weight fraction of silane coupling agent using scanning electron microscopy (SEM), a four-point probe method and adhesion testing. In addition, a bending fatigue test was performed to evaluate the reliability of the conductive copper pattern under cyclic bending. The copper pattern fabricated with copper complex ion ink containing 3 wt% silane coupling agent exhibited the highest adhesion level (5B), the lowest resistivity (7.6 μΩ·cm) and a low resistance change (18%) after the bending fatigue test. The two-step sintering method used to enhance the adhesion between the copper complex ion ink and polyimide substrate was also studied using X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR). - Highlights: • The copper complex ion ink with copper nanoparticles and copper precursor was fabricated. • The copper complex ion ink was sintered by two-step flash light sintering method. • The sintered copper pattern exhibited the highest adhesion level (5B). • The resistivity of sintered copper pattern was 7.6 μΩ·cm.

  2. Distant polypharmacology among MLP chemical probes.

    Science.gov (United States)

    Antolín, Albert A; Mestres, Jordi

    2015-02-20

    Small molecules are essential tool compounds to probe the role of proteins in biology and advance toward more efficient therapeutics. However, they are used without a complete knowledge of their selectivity across the entire proteome, at risk of confounding their effects due to unknown off-target interactions. Current state-of-the-art computational approaches to predicting the affinity profile of small molecules offer a means to anticipate potential nonobvious selectivity liabilities of chemical probes. The application of in silico target profiling on the full set of chemical probes from the NIH Molecular Libraries Program (MLP) resulted in the identification of biologically relevant in vitro affinities for proteins distantly related to the primary targets of ML006, ML123, ML141, and ML204, helping to lower the risk of their further use in chemical biology.

  3. Copper (II) complexes with aroylhydrazones

    Indian Academy of Sciences (India)

    Copper(II) complexes with aroylhydrazones ... The coordination chemistry of copper(II) with tridentate aroylhydrazones is briefly discussed in this article. ... EPR spectroscopy and variable temperature magnetic susceptibility measurements have been used to reveal the nature of the coordination geometry and magnetic ...

  4. The Bauschinger Effect in Copper

    DEFF Research Database (Denmark)

    Pedersen, Ole Bøcker; Brown, L .M.; Stobbs, W. M.

    1981-01-01

    A study of the Bauschinger effect in pure copper shows that by comparison with dispersion hardened copper the effect is very small and independent of temperature. This suggests that the obstacles to flow are deformable. A simple composite model based on this principle accounts for the data semi...

  5. Copper nanoparticle modified carbon electrode for determination of dopamine

    International Nuclear Information System (INIS)

    Oztekin, Yasemin; Tok, Mutahire; Bilici, Esra; Mikoliunaite, Lina; Yazicigil, Zafer; Ramanaviciene, Almira; Ramanavicius, Arunas

    2012-01-01

    This paper reports the synthesis and characterization of copper nanoparticles (CuNPs) and application of copper nanoparticle-modified glassy carbon electrode for the electrochemical determination of dopamine. Electrochemical measurements were performed using differently modified glassy carbon (GC) electrodes. Bare, oxidized before modification and copper nanoparticle-modified glassy carbon electrodes (bare-GC, ox-GC and CuNP/GC electrodes, respectively) were characterized by cyclic voltammetry and electrochemical impedance spectroscopy in the presence of redox probes. Atomic force microscopy was used for the visualization of electrode surfaces. The CuNP/GC electrode was found to be suitable for the selective determination of dopamine even in the presence of ascorbic acid, uric acid, and p-acetamidophenol. The observed linear range of CuNP/GC for dopamine was from 0.1 nM to 1.0 μM while the detection limit was estimated to be 50 pM. It was demonstrated that here reported glassy carbon electrode modified by copper nanoparticles is suitable for the determination of dopamine in real samples such as human blood serum.

  6. Atom probe tomography analysis of WC powder

    Energy Technology Data Exchange (ETDEWEB)

    Weidow, Jonathan, E-mail: jonathan.weidow@chalmers.se [Department of Applied Physics, Chalmers University of Technology, SE-412 96 Göteborg (Sweden); Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Wien (Austria)

    2013-09-15

    A tantalum doped tungsten carbide powder, (W,Ta)C, was prepared with the purpose to maximise the amount of Ta in the hexagonal mixed crystal carbide. Atom probe tomography (APT) was considered to be the best technique to quantitatively measure the amount of Ta within this carbide. As the carbide powder consisted in the form of very small particles (<1 μm), a method to produce APT specimens of such a powder was developed. The powder was at first embedded in copper and a FIB-SEM workstation was used to make an in-situ lift-out from a selected powder particle. The powder particle was then deposited on a post made from a WC-Co based cemented carbide specimen. With the use of a laser assisted atom probe, it was shown that the method is working and the Ta content of the (W,Ta)C could be measured quantitatively. - Highlights: ► Method for producing atom probe tomography specimens of powders was developed. ► Method was successfully implemented on (W,Ta)C powder. ► Method can possibly be implemented on completely other powders.

  7. Potential use of copper as a hygienic surface; problems associated with cumulative soiling and cleaning.

    Science.gov (United States)

    Airey, P; Verran, J

    2007-11-01

    It has been suggested that antibacterial copper could be used in place of stainless steel to help reduce the occurrence of hospital-acquired infections. The antibacterial activity of copper has been clearly demonstrated when using cell suspensions held in prolonged contact with copper or copper alloys. The aim of this study was to evaluate the antimicrobial properties of copper in comparison with stainless steel in a generally dry environment. Three stainless steels of varying surface finish and polished copper were soiled with Staphylococcus aureus suspended in a protein-based organic soil (bovine serum album), dried rapidly, and then incubated for 24 h. Surfaces were then wiped clean using a standardised wiping procedure with two cleaning agents recommended by UK National Health Service guidelines. This soiling/cleaning procedure was carried out daily over five days. After each cleaning cycle the amount of residual soil and live cells was assessed using direct epifluorescence microscopy. All materials were easily cleaned after the first soiling episode but a build-up of cells and soil was observed on the copper surfaces after several cleaning/wiping cycles. Stainless steel remained highly cleanable. Accumulation of material on copper is presumably due to the high reactivity of copper, resulting in surface conditioning. This phenomenon will affect subsequent cleaning, aesthetic properties and possibly antibacterial performance. It is important to select the appropriate cleaning/disinfecting protocols for selected surfaces.

  8. Identification of Two Conserved Residues Involved in Copper Release from Chloroplast PIB-1-ATPases.

    Science.gov (United States)

    Sautron, Emeline; Giustini, Cécile; Dang, ThuyVan; Moyet, Lucas; Salvi, Daniel; Crouzy, Serge; Rolland, Norbert; Catty, Patrice; Seigneurin-Berny, Daphné

    2016-09-16

    Copper is an essential transition metal for living organisms. In the plant model Arabidopsis thaliana, half of the copper content is localized in the chloroplast, and as a cofactor of plastocyanin, copper is essential for photosynthesis. Within the chloroplast, copper delivery to plastocyanin involves two transporters of the PIB-1-ATPases subfamily: HMA6 at the chloroplast envelope and HMA8 in the thylakoid membranes. Both proteins are high affinity copper transporters but share distinct enzymatic properties. In the present work, the comparison of 140 sequences of PIB-1-ATPases revealed a conserved region unusually rich in histidine and cysteine residues in the TMA-L1 region of eukaryotic chloroplast copper ATPases. To evaluate the role of these residues, we mutated them in HMA6 and HMA8. Mutants of interest were selected from phenotypic tests in yeast and produced in Lactococcus lactis for further biochemical characterizations using phosphorylation assays from ATP and Pi Combining functional and structural data, we highlight the importance of the cysteine and the first histidine of the CX3HX2H motif in the process of copper release from HMA6 and HMA8 and propose a copper pathway through the membrane domain of these transporters. Finally, our work suggests a more general role of the histidine residue in the transport of copper by PIB-1-ATPases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Combining -Omics to Unravel the Impact of Copper Nutrition on Alfalfa (Medicago sativa) Stem Metabolism.

    Science.gov (United States)

    Printz, Bruno; Guerriero, Gea; Sergeant, Kjell; Audinot, Jean-Nicolas; Guignard, Cédric; Renaut, Jenny; Lutts, Stanley; Hausman, Jean-Francois

    2016-02-01

    Copper can be found in the environment at concentrations ranging from a shortage up to the threshold of toxicity for plants, with optimal growth conditions situated in between. The plant stem plays a central role in transferring and distributing minerals, water and other solutes throughout the plant. In this study, alfalfa is exposed to different levels of copper availability, from deficiency to slight excess, and the impact on the metabolism of the stem is assessed by a non-targeted proteomics study and by the expression analysis of key genes controlling plant stem development. Under copper deficiency, the plant stem accumulates specific copper chaperones, the expression of genes involved in stem development is decreased and the concentrations of zinc and molybdenum are increased in comparison with the optimum copper level. At the optimal copper level, the expression of cell wall-related genes increases and proteins playing a role in cell wall deposition and in methionine metabolism accumulate, whereas copper excess imposes a reduction in the concentration of iron in the stem and a reduced abundance of ferritins. Secondary ion mass spectrometry (SIMS) analysis suggests a role for the apoplasm as a copper storage site in the case of copper toxicity. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  10. Copper reverses cardiomyocyte hypertrophy through vascular endothelial growth factor-mediated reduction in the cell size.

    Science.gov (United States)

    Zhou, Yang; Jiang, Youchun; Kang, Y James

    2008-07-01

    Previous studies have shown that dietary copper supplementation reversed heart hypertrophy induced by pressure overload in a mouse model. The present study was undertaken to understand the cellular basis of copper-induced regression of cardiac hypertrophy. Primary cultures of neonatal rat cardiomyocytes were treated with phenylephrine (PE) at a final concentration of 100 microM in cultures for 48 h to induce cellular hypertrophy. The hypertrophied cardiomyocytes were exposed to copper sulfate at a final concentration of 5 microM in cultures for additional 24 h. This copper treatment reduced the size of the hypertrophied cardiomyocytes, as measured by flow cytometry, protein content in cells, cell volume and cardiomyocyte hypertrophy markers including beta-myosin heavy chain protein, skeletal alpha-actin, and atrial natriuretic peptide. Cell cycle analysis and cell sorting of p-histone-3 labeled cardiomyocytes indicated that cell division was not involved in the copper-induced regression of cardiomyocyte hypertrophy. Copper also inhibited PE-induced apoptosis, determined by a TUNEL assay. Because copper stimulates vascular endothelial growth factor (VEGF) production through activation of hypoxia-inducible transcription factor, an anti-VEGF antibody at a final concentration of 2 ng/ml in cultures was used and shown to blunt copper-induced regression of cell hypertrophy. Conversely, VEGF alone at a final concentration of 0.2 microg/ml reversed cell hypertrophy as the same as copper did. This study demonstrates that both copper and VEGF reduce the size of hypertrophied cardiomyocytes, and copper regression of cardiac hypertrophy is VEGF-dependent.

  11. Sublethal concentrations of waterborne copper induce cellular stress and cell death in zebrafish embryos and larvae

    Directory of Open Access Journals (Sweden)

    Pedro P Hernandez

    2011-01-01

    Full Text Available Copper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fish. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fish. Here, acute toxicity tests and LC50 curves have been generated for zebrafish larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentration-dependent manner, resulting in changes in gene expression. Using a transgenic fish line that expresses enhanced green fluorescent protein (EGFP under the hsp70 promoter, we monitored tissue-specific stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals.

  12. The involvement of vimentin in copper-induced regression of cardiomyocyte hypertrophy.

    Science.gov (United States)

    Li, Rui; Bourcy, Katherine; Wang, Tao; Sun, Miao; Kang, Y James

    2015-09-01

    Dietary copper supplementation reverses the pressure overload-induced cardiac hypertrophy. Activation of vascular endothelial growth factor receptor-1 (VEGFR-1) and cyclic guanosine monophosphate (cGMP)-dependent protein kinase-1 (PKG-1) is required for the regression. The present study was undertaken to determine the link between VEGFR-1 and PKG-1 in copper regression of cardiomyocyte hypertrophy. Human cardiac myocytes (HCM) or primary cultures of neonatal rat cardiomyocytes were exposed to phenylephrine (PE) at a final concentration of 100 μM for 48 h to induce cell hypertrophy. Copper sulfite was added to cultures of hypertrophic cardiomyocytes at a final concentration of 5 μM elemental copper and incubated for 24 h to reverse cell hypertrophy. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified a 56 kDa copper-binding protein, vimentin, which was co-immunoprecipitated with VEGFR-1 and PKG-1. Copper supplementation increased vimentin levels and enhanced PKG-1 activity. Gene silencing using siRNA targeting vimentin prevented copper-induced elevation of vimentin, depressed the activity of PKG-1, and blocked the copper-induced regression of cardiomyocyte hypertrophy. This study demonstrates that vimentin is critically involved in the VEGFR-1 mediated activation of the PKG-1 signaling pathway, leading to regression of cardiomyocyte hypertrophy.

  13. The Clp protease system is required for copper ion-dependent turnover of the PAA2/HMA8 copper transporter in chloroplasts.

    Science.gov (United States)

    Tapken, Wiebke; Kim, Jitae; Nishimura, Kenji; van Wijk, Klaas J; Pilon, Marinus

    2015-01-01

    The distribution of essential metal ions over subcellular compartments for use as cofactors requires control of membrane transporters. PAA2/HMA8 is a copper-transporting P1B -type ATPase in the thylakoid membrane, required for the maturation of plastocyanin. When copper is highly available to the plant this transporter is degraded, which implies the action of a protease. In order to identify the proteolytic machinery responsible for PAA2/HMA8 turnover in Arabidopsis, mutant lines defective in five different chloroplast protease systems were analyzed. Plants defective in the chloroplast caseinolytic protease (Clp) system were specifically impaired in PAA2/HMA8 protein turnover on media containing elevated copper concentrations. However, the abundance of a core Clp component was not directly affected by copper. Furthermore, the expression and activity of both cytosolic and chloroplast-localized superoxide dismutases (SODs), which are known to be dependent on copper, were not altered in the clp mutants, indicating that the loss of PAA2/HMA8 turnover in these lines was not caused by a lack of stromal copper. The results suggest that copper excess in the stroma triggers selection of the thylakoid-localized PAA2 transporter for degradation by the Clp protease, but not several other chloroplast proteases, and support a novel role for this proteolytic system in cellular copper homeostasis. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  14. Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase.

    Science.gov (United States)

    Liu, Jingxuan; Sitaram, Anand; Burd, Christopher G

    2007-10-01

    The Saccharomyces cerevisiae high-affinity copper transporter, Ctr1p, mediates cellular uptake of Cu(I). We report that when copper (50 microm CuSO(4)) is added to the growth medium of copper-starved cells, Ctr1p is rapidly internalized by endocytosis, delivered to the lumen of the lysosome-like vacuole and slowly degraded by vacuolar proteases. Through analysis of the trafficking and degradation of Ctr1p mutants, two lysine residues in the C-terminal cytoplasmic tail of Ctr1p, Lys340 and Lys345, were found to be critical for copper-dependent endocytosis and degradation. In response to copper addition, Ctr1p was found to be ubiquitylated and a mutation in the Rsp5 ubiquitin ligase largely abolished ubiquitylation, endocytosis and degradation. In a strain lacking the Rsp5p accessory factors Bul1p and Bul2p, endocytosis and degradation of Ctr1p-green fluorescent protein were substantially diminished. Surprisingly, a Ctr1p mutant that lacks Lys340 and Lys345 was still ubiquitylated in a copper-dependent manner, indicating that ubiquitylation of Ctr1p on other sites is insufficient to drive copper-dependent endocytosis and degradation. This study demonstrates that copper regulates turnover of Ctr1p by stimulating Rsp5p-dependent endocytosis and degradation of Ctr1p in the vacuole.

  15. Ultrasonic Inspection Techniques Possibilities for Centrifugal Cast Copper Alloy

    Directory of Open Access Journals (Sweden)

    Konar R.

    2017-06-01

    Full Text Available The article deals with ultrasonic testing possibilities of the copper alloy centrifugal casts. It focused on the problems that arise when testing of castings is made of non-ferrous materials. Most common types of casting defects is dedicated in theoretical introduction of article. Ultrasonic testing technique by conventional ultrasound system is described in the theoretical part too. Practical ultrasonic testing of centrifugal copper alloy cast - brass is in experimental part. The experimental sample was part of centrifugally cast brass ring with dimensions of Ø1200x34 mm. The influence of microstructure on ultrasonic attenuation and limitations in testing due to attenuation is describes in experimental part. Conventional direct single element contact ultrasound probe with frequencies of 5 MHz, 3.5 MHz and 2 MHz were used for all experimental measurements. The results of experimental part of article are recommendations for selecting equipment and accessories for casting testing made of non-ferrous metals.

  16. The role of insufficient copper in lipid synthesis and fatty-liver disease.

    Science.gov (United States)

    Morrell, Austin; Tallino, Savannah; Yu, Lei; Burkhead, Jason L

    2017-04-01

    The essential transition metal copper is important in lipid metabolism, redox balance, iron mobilization, and many other critical processes in eukaryotic organisms. Genetic diseases where copper homeostasis is disrupted, including Menkes disease and Wilson disease, indicate the importance of copper balance to human health. The severe consequences of insufficient copper supply are illustrated by Menkes disease, caused by mutation in the X-linked ATP7A gene encoding a protein that transports copper from intestinal epithelia into the bloodstream and across the blood-brain barrier. Inadequate copper supply to the body due to poor diet quality or malabsorption can disrupt several molecular level pathways and processes. Though much of the copper distribution machinery has been described and consequences of disrupted copper handling have been characterized in human disease as well as animal models, physiological consequences of sub-optimal copper due to poor nutrition or malabsorption have not been extensively studied. Recent work indicates that insufficient copper may be important in a number of common diseases including obesity, ischemic heart disease, and metabolic syndrome. Specifically, marginal copper deficiency (CuD) has been reported as a potential etiologic factor in diseases characterized by disrupted lipid metabolism such as non-alcoholic fatty-liver disease (NAFLD). In this review, we discuss the available data suggesting that a significant portion of the North American population may consume insufficient copper, the potential mechanisms by which CuD may promote lipid biosynthesis, and the interaction between CuD and dietary fructose in the etiology of NAFLD. © 2016 IUBMB Life, 69(4):263-270, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  17. Copper uptake and retention in liver parenchymal cells isolated from nutritionally copper-deficient rats

    NARCIS (Netherlands)

    Berg, G.J. van den; Goeij, J.J.M. de; Bock, I.; Gijbels, M.J.J.; Brouwer, A.; Lei, K.Y.; Hendruiks, H.F.J.

    1991-01-01

    Copper uptake and retention were studied in primary cultures of liver parenchymal cells isolated from copper-deficient rats. Male Sprague-Dawley rats were fed a copper-deficient diet (< 1 mg Cu/kg) for 10 wk. Copper-deficient rats were characterized by low copper concentrations in plasma and liver,

  18. Neutrons as a probe

    International Nuclear Information System (INIS)

    Iizumi, Masashi

    1993-01-01

    As an introduction to the symposium a brief overview will be given about the features of neutrons as a probe. First it will be pointed out that the utilization of neutrons as a probe for investigating the structural and dynamical properties of condensed matters is a benign gift eventuated from the release of atomic energy initiated by Enrico Fermi exactly half century ago. Features of neutrons as a probe are discussed in accordance with the four basic physical properties of neutrons as an elementary particle; (1) no electric charge (the interaction with matter is nuclear), (2) the mass of neutron is 1 amu, (3) spin is 1/2 and (4) neutrons have magnetic dipole moment. Overview will be given on the uniqueness of neutrons as a probe and on the variety in the way they are used in the wide research area from the pure science to the industrial applications. (author)

  19. Copper complexes as 'radiation recovery' agents

    International Nuclear Information System (INIS)

    Sorenson, J.R.J.

    1989-01-01

    Copper and its compounds have been used for their remedial effects since the beginning of recorded history. As early as 3000 BC the Egyptians used copper as an antiseptic for healing wounds and to sterilise drinking water; and later, ca 1550 BC, the Ebers Papyrus reports the use of copper acetate, copper sulphate and pulverised metallic copper for the treatment of eye infections. These historical uses of copper and its compounds are particularly interesting in the light of modern evidence concerning the use of certain copper complexes for the treatment of radiation sickness and more recently as an adjunct to radiotherapy for cancer patients. (author)

  20. NID Copper Sample Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Kouzes, Richard T.; Zhu, Zihua

    2011-09-12

    The current focal point of the nuclear physics program at PNNL is the MAJORANA DEMONSTRATOR, and the follow-on Tonne-Scale experiment, a large array of ultra-low background high-purity germanium detectors, enriched in 76Ge, designed to search for zero-neutrino double-beta decay (0νββ). This experiment requires the use of germanium isotopically enriched in 76Ge. The MAJORANA DEMONSTRATOR is a DOE and NSF funded project with a major science impact. The DEMONSTRATOR will utilize 76Ge from Russia, but for the Tonne-Scale experiment it is hoped that an alternate technology, possibly one under development at Nonlinear Ion Dynamics (NID), will be a viable, US-based, lower-cost source of separated material. Samples of separated material from NID require analysis to determine the isotopic distribution and impurities. DOE is funding NID through an SBIR grant for development of their separation technology for application to the Tonne-Scale experiment. The Environmental Molecular Sciences facility (EMSL), a DOE user facility at PNNL, has the required mass spectroscopy instruments for making isotopic measurements that are essential to the quality assurance for the MAJORANA DEMONSTRATOR and for the development of the future separation technology required for the Tonne-Scale experiment. A sample of isotopically separated copper was provided by NID to PNNL in January 2011 for isotopic analysis as a test of the NID technology. The results of that analysis are reported here. A second sample of isotopically separated copper was provided by NID to PNNL in August 2011 for isotopic analysis as a test of the NID technology. The results of that analysis are also reported here.

  1. Gallium and copper radiopharmaceutical chemistry

    International Nuclear Information System (INIS)

    Green, M.A.

    1991-01-01

    Gallium and copper radionuclides have a long history of use in nuclear medicine. Table 1 presents the nuclear properties of several gallium and copper isotopes that either are used in the routine practice of clinical nuclear medicine or exhibit particular characteristics that might make them useful in diagnostic or therapeutic medicine. This paper will provide some historic perspective along with an overview of some current research directions in gallium and copper radiopharmaceutical chemistry. A more extensive review of gallium radiopharmaceutical chemistry has recently appeared and can be consulted for a more in-depth treatment of this topic

  2. Control of biofouling on titanium condenser tubes with the use of electroless copper plating

    International Nuclear Information System (INIS)

    Anandkumar, B.; George, R.P.; Kamachi Mudali, U.; Ramachandran, D.

    2015-01-01

    In sea water environments titanium condenser tubes face serious issues of biofouling and biomineralization. Electroless plating of nanocopper film is attempted inside the tubes for the control of biofilm formation. Using advanced techniques like AFM, SEM, and XPS, electroless copper plated flat Ti specimens were characterized. Examination of Cu coated Ti surfaces using AFM and SEM showed more reduction in the microroughness compared to anodized Ti surface. Cu 2p 3/2 peak in XPS spectral analysis showed the shift in binding energy inferring the reduction of the hydroxide to metallic copper. Tubular specimens were exposed to sea water up to three months and withdrawn at monthly intervals to evaluate antibacterial activity and long term stability of the coating. Total viable counts and epifluorescence microscopy analyses showed two orders decrease in bacterial counts on copper coated Ti specimens when compared to as polished control Ti specimens. Molecular biology techniques like DGGE and protein expression analysis system were done to get insight into the community diversity and copper tolerance of microorganisms. DGGE gel bands clearly showed the difference in the bacterial diversity inferring from the 16S rRNA gene fragments (V3 regions). Protein analysis showed distinct protein spots appearing in electroless copper coated Ti biofilm protein samples in addition to protein spots common to both the biofilms of Cu coated and as polished Ti. The results indicated copper accumulating proteins in copper resistant bacterial species of biofilm. Reduced microroughness of the surface and toxic copper ions resulted in good biofouling control even after three months exposure to sea water. (author)

  3. A colorimetric probe for the detection of Ni2+ in water based on Ag ...

    African Journals Online (AJOL)

    A Ni2+ based colorimetric probe based on glutathione-stabilized silver/copper nanoparticles (GSH-Ag-Cu alloy NPs) in an electrospun polymer matrix is reported. Glutathione-Ag-Cu alloy NPs were characterized by ultraviolet-visible spectroscopy (UV-vis), scanning electron microscopy (SEM) and transmission electron ...

  4. Development of sensors, probes and imaging techniques for pollutant monitoring in geo-environmental model tests

    NARCIS (Netherlands)

    Lynch, R.J.; Allersma, H.; Barker, H.; Bezuijen, A.; Bolton, M.D.; Cartwright, M.; Davies, M.C.R.; Depountis, N.; Esposito, G.; Garnier, J.; Almeida Garrett, J.L.L. de; Harris, C.; Kechavarzi, C.; Oung, O.; Silva, M.A.G. da; Santos, C.; Sentenac, P.; Soga, K.; Spiessl, S.; Taylor, R.N.; Treadaway, A.C.J.; Weststrate, F.

    2001-01-01

    In order to be able to track the movement of pollutant plumes during geotechnical centrifuge and other geo-en-vironmental experiments, a number of techniques have been investigated: fibre-optic photometric sensors, resistivity probes, resistivity tomography, and copper ion-selective electrodes.

  5. Noninvasive ultrasonic probes

    International Nuclear Information System (INIS)

    Barnes, S.R.; Galer, D.R.; Leard, R.S.

    1987-01-01

    An ultrasonic probe is described for insonifying the ascending aorta of a supine or reclining human patient from a location within the suprasternal notch of the patient. The probe comprises: a transducer head and an elongated handle; housing propagates ultrasonic energy and for intercept-frequency-shifted, reflected radiant energy; the handle has a proximate portion and a distal portion and a non-circular cross-sectional configuration with at least one longitudinal edge which furnishes a gripping surface; this facilitates tactile positioning of the probe; the transducer head is integral with the handle of the probe at the exposed end of the proximate portion; the transducer head has a generally arcuate cross-sectional configuration and a generally trapezoidal profile; the transducer head is oriented at right angles to the proximate portion of the handle and has an exposed, patient contacting end in which the transducer means are located; this facilitates the orientation of the transducer means housed in the head relative to the ascending aorta of the patient; and the distal end portion of the elongated probe handle is integral with and immovably oriented at a severe angle relative to the proximate end of that handle, and lies in the same plane as the proximate end of the handle; the transducer head of the probe is placed with facility within the suprasternal notch of the patient by an operator positioned behind the head of the patient

  6. Rapid deuterium exchange-in time for probing conformational change

    International Nuclear Information System (INIS)

    Dharmasiri, K.; Smith, D.L.

    1995-01-01

    Isotopic exchange of protein backbone amide hydrogens has been used extensively as a sensitive probe of protein structure. One of the salient features of hydrogen exchange is the vast range of exchange rates in one protein. Isotopic exchange methods have been used to study the structural features including protein folding and unfolding (1), functionally different forms of proteins (2), protein-protein complexation (3), and protein stability parameter. Many backbone amide protons that are surface accessible and are not involved in hydrogen bonding undergo rapid deuterium exchange. In order to study, fast exchanging amide protons, fast exchange-in times are necessary

  7. Chemical Probes of Histone Lysine Methyltransferases

    Science.gov (United States)

    2015-01-01

    Growing evidence suggests that histone methyltransferases (HMTs, also known as protein methyltransferases (PMTs)) play an important role in diverse biological processes and human diseases by regulating gene expression and the chromatin state. Therefore, HMTs have been increasingly recognized by the biomedical community as a class of potential therapeutic targets. High quality chemical probes of HMTs, as tools for deciphering their physiological functions and roles in human diseases and testing therapeutic hypotheses, are critical for advancing this promising field. In this review, we focus on the discovery, characterization, and biological applications of chemical probes for HMTs. PMID:25423077

  8. Modulation of lysyl oxidase by dietary copper in rats.

    Science.gov (United States)

    Rucker, R B; Romero-Chapman, N; Wong, T; Lee, J; Steinberg, F M; McGee, C; Clegg, M S; Reiser, K; Kosonen, T; Uriu-Hare, J Y; Murphy, J; Keen, C L

    1996-01-01

    Lysyl oxidase levels were estimated in rat tissues using an enzyme-linked immunosorption assay (ELISA) and a functional assay standardized against known amounts of purified lysyl oxidase. High concentrations of lysyl oxidase (> or = 150 micrograms/g of tissue or packed cells) were detected in connective tissues, such as tendon and skin. Values for aorta, kidney, lung and liver ranged from 30 to 150 micrograms/g of tissue; values for skeletal muscle and diaphragm were tendon (r2 > 0.9). When egg white-based experimental diets containing 2 or 10 micrograms/g added copper were fed to weanling rats, values for skin lysyl oxidase functional activity in the group fed 2 micrograms/g added copper were one-third to one-half the values for skin lysyl oxidase functional activity in rats fed 10 micrograms/g copper. This reduction in lysyl oxidase activity, however, had minimal effect on indices of collagen maturation in rat skin, e.g., collagen solubility in neutral salt and dilute acid or the levels of acid stable cross-links. Moreover, copper deficiency did not influence the steady-state levels of lysyl oxidase specific mRNA in rat skin or the apparent amounts of lysyl oxidase in rat skin as determined by ELISA. These observations underscore that the concentration of lysyl oxidase is relatively high in dense corrective tissues, and although decreasing dietary copper influences functional activity, there is little apparent effect on the production of lysyl oxidase protein.

  9. Differential sexual survival of Drosophila melanogaster on copper sulfate.

    Science.gov (United States)

    Balinski, Michael A; Woodruff, Ronny C

    2017-04-01

    Based on studies of the influence of X-chromosomes on the viability of Drosophila melanogaster exposed to cadmium, and on the role of X-linked genes on copper homeostasis, we examined the effect of copper sulfate (CuSO 4 ) on offspring viability using three independent, inbred D. melanogaster crosses (ensuring identical autosomes for males and females within each cross). Each cross was performed with attached X-chromosome females and males with a single X-chromosome. As female D. melanogaster have less metallothionein RNA expression than males, we predicted fewer female offspring than male offspring in crosses exposed to CuSO 4 , even though females have two copies of X-chromosome genes, possibly resulting in overdominant heterozygosity. In two of three crosses, CuSO 4 caused significantly higher numbers of male offspring compared to female offspring. We hypothesized that these gender-based viability differences to copper exposure are caused by X-chromosome ploidy and X-linked genetic variation affecting metallothionein expression. Observed differential offspring viability responses among crosses to copper exposure also showed that different genetic backgrounds (autosomal and/or X-chromosome) can result in significant differences in heavy metal and metallothionein regulation. These results suggest that the effect of copper on offspring viability depends on both genetic background and gender, as both factors can affect the regulation of metallothionein proteins as well as homeostasis of biologically necessary heavy metals.

  10. Phosphorylated α-Synuclein-Copper Complex Formation in the Pathogenesis of Parkinson’s Disease

    Directory of Open Access Journals (Sweden)

    Juan Antonio Castillo-Gonzalez

    2017-01-01

    Full Text Available Parkinson’s disease is the second most important neurodegenerative disorder worldwide. It is characterized by the presence of Lewy bodies, which are mainly composed of α-synuclein and ubiquitin-bound proteins. Both the ubiquitin proteasome system (UPS and autophagy-lysosomal pathway (ALS are altered in Parkinson’s disease, leading to aggregation of proteins, particularly α-synuclein. Interestingly, it has been observed that copper promotes the protein aggregation process. Additionally, phosphorylation of α-synuclein along with copper also affects the protein aggregation process. The interrelation among α-synuclein phosphorylation and its capability to interact with copper, with the subsequent disruption of the protein degradation systems in the neurodegenerative process of Parkinson’s disease, will be analyzed in detail in this review.

  11. Copper transfer from Cu-Aβ to human serum albumin inhibits aggregation, radical production and reduces Aβ toxicity

    DEFF Research Database (Denmark)

    Perrone, Lorena; Mothes, Emmanuelle; Vignes, Maeva

    2010-01-01

    Amyloid-β peptides (Aβ) and the protein human serum albumin (HSA) interact in vivo. They are both localised in the blood plasma and in the cerebrospinal fluid. Among other functions, HSA is involved in the transport of the essential metal copper. Complexes between Aβ and copper ions have been...

  12. Copper tailings in stucco mortars

    Directory of Open Access Journals (Sweden)

    Osvaldo Pavez

    Full Text Available Abstract This investigation addressed the evaluation of the use of copper tailings in the construction industry in order to reduce the impact on the environment. The evaluation was performed by a technical comparison between stucco mortars prepared with crushed conventional sand and with copper tailings sand. The best results were achieved with the stucco mortars containing tailings. The tailings presented a fine particles size distribution curve different from that suggested by the standard. The values of compressive strength, retentivity, and adherence in the stucco mortars prepared with copper tailings were much higher than those obtained with crushed sand. According to the results from this study, it can be concluded that the preparation of stucco mortars using copper tailings replacing conventional sand is a technically feasible alternative for the construction industry, presenting the benefit of mitigating the impact of disposal to the environment.

  13. Hereditary iron and copper deposition

    DEFF Research Database (Denmark)

    Aaseth, Jan; Flaten, Trond Peder; Andersen, Ole

    2007-01-01

    Hereditary deposition of iron (primary haemochromatosis) or copper (Wilson's disease) are autosomal recessive metabolic disease characterized by progressive liver pathology and subsequent involvement of various other organs. The prevalence of primary haemochromatosis is approximately 0.5%, about...

  14. Thermal Stability of Copper-Aluminum Alloy Thin Films for Barrierless Copper Metallization on Silicon Substrate

    Science.gov (United States)

    Wang, C. P.; Dai, T.; Lu, Y.; Shi, Z.; Ruan, J. J.; Guo, Y. H.; Liu, X. J.

    2017-08-01

    Copper thin films with thickness of about 500 nm doped with different aluminum concentrations have been prepared by magnetron sputtering on Si substrate and their crystal structure, microstructure, and electrical resistivity after annealing at various temperatures (200°C to 600°C) for 1 h or at 400°C for different durations (1 h to 11 h) investigated by grazing-incidence x-ray diffraction (GIXRD) analysis, scanning electron microscopy (SEM), and four-point probe (FPP) measurements. Cu-1.8Al alloy thin film exhibited good thermal stability and low electrical resistivity (˜5.0 μΩ cm) after annealing at 500°C for 1 h or 400°C for 7 h. No copper silicide was observed at the Cu-Al/Si interface by GIXRD analysis or SEM for this sample. This result indicates that doping Cu thin film with small amounts of Al can achieve high thermal stability and low electrical resistivity, suggesting that Cu-1.8Al alloy thin film could be used for barrierless Cu metallization on Si substrate.

  15. Distribution and chemical forms of copper in the root cells of castor seedlings and their tolerance to copper phytotoxicity in hydroponic culture.

    Science.gov (United States)

    Kang, Wei; Bao, Jianguo; Zheng, Jin; Hu, Hongqin; Du, Jiangkun

    2015-05-01

    The subcellular localization and chemical forms of copper in castor (Ricinus communis L.) seedlings grown in hydroponic nutrient solution were identified by chemical extraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The wild castor seeds were harvested from an abandoned copper mine in Tonglu Mountain, Daye City of Hubei Province, China. The results revealed that (1) the seedlings grew naturally in MS liquid medium with 40.00 mg kg(-1) CuSO4, in which the seedling growth rate and biomass index were 0.14 and 1.23, respectively, which were the highest values among all the treatments. The copper content in castor seedlings increased along with elevated CuSO4 concentration in the medium, reaching a maximum value of 16 570.12 mg kg(-1)(DW) when exposed to 60.00 mg L(-1) CuSO4, where 91.31% of the copper was accumulated in roots. (2) The copper existed in various chemical forms in the roots of the castor seedlings. Copper of 67.66% was extracted from the components of cell walls, such as exchangeable acidic polar compounds, cellulose and lignin, protein and pectin, and less concentrated in cell cytoplasm and nuclei. (3) Furthermore, the root cell walls were thickened when the castor seedlings exposed to CuSO4, with a large amount of high-density electron bodies, attached to the thickened cell walls. In the cell walls, most copper was bound to the carboxyl (-COOH) and hydroxyl (-OH) groups of acidic polar compounds, cellulose, hemicellulose, and polysaccharides. The conclusion showed that castor exhibited a strong tolerance to copper, the copper were accumulated mainly in the root cell, the root cell walls of castor were the major location of patience and detoxification in copper stress.

  16. Copper atomic-scale transistors

    Directory of Open Access Journals (Sweden)

    Fangqing Xie

    2017-03-01

    Full Text Available We investigated copper as a working material for metallic atomic-scale transistors and confirmed that copper atomic-scale transistors can be fabricated and operated electrochemically in a copper electrolyte (CuSO4 + H2SO4 in bi-distilled water under ambient conditions with three microelectrodes (source, drain and gate. The electrochemical switching-on potential of the atomic-scale transistor is below 350 mV, and the switching-off potential is between 0 and −170 mV. The switching-on current is above 1 μA, which is compatible with semiconductor transistor devices. Both sign and amplitude of the voltage applied across the source and drain electrodes (Ubias influence the switching rate of the transistor and the copper deposition on the electrodes, and correspondingly shift the electrochemical operation potential. The copper atomic-scale transistors can be switched using a function generator without a computer-controlled feedback switching mechanism. The copper atomic-scale transistors, with only one or two atoms at the narrowest constriction, were realized to switch between 0 and 1G0 (G0 = 2e2/h; with e being the electron charge, and h being Planck’s constant or 2G0 by the function generator. The switching rate can reach up to 10 Hz. The copper atomic-scale transistor demonstrates volatile/non-volatile dual functionalities. Such an optimal merging of the logic with memory may open a perspective for processor-in-memory and logic-in-memory architectures, using copper as an alternative working material besides silver for fully metallic atomic-scale transistors.

  17. The copper deposits of Michigan

    Science.gov (United States)

    Butler, B.S.; Burbank, W.S.

    1929-01-01

    The copper district of Keweenaw Point, in the northern peninsula of Michigan, is the second largest producer of copper in the world.  The output of the district since 1845 has been more than 7,500,000,000 pounds and showed a rather steady and consistent increase from the beginning of production to the end of the World War in 1918, since which there has been a marked decrease.

  18. Atmospheric corrosion effects on copper

    International Nuclear Information System (INIS)

    Franey, J.P.

    1985-01-01

    Studies have been performed on the naturally formed patina on various copper samples. Samples have been obtained from structures at AT and T Bell Laboratories, Murray Hill, NJ (40,2,1 and <1 yr) and the Statue of Liberty (100 yr). The samples show a distinct layering effect, that is, the copper base material shows separate oxide and basic sulfate layers on all samples, indicating that patina is not a homogeneous mixture of oxides and basic sulfates

  19. Model for resonant plasma probe.

    Energy Technology Data Exchange (ETDEWEB)

    Warne, Larry Kevin; Johnson, William Arthur; Hebner, Gregory Albert; Jorgenson, Roy E.; Coats, Rebecca Sue

    2007-04-01

    This report constructs simple circuit models for a hairpin shaped resonant plasma probe. Effects of the plasma sheath region surrounding the wires making up the probe are determined. Electromagnetic simulations of the probe are compared to the circuit model results. The perturbing effects of the disc cavity in which the probe operates are also found.

  20. Genome Sequences of Two Copper-Resistant Escherichia coli Strains Isolated from Copper-Fed Pigs

    DEFF Research Database (Denmark)

    Lüthje, Freja L.; Hasman, Henrik; Aarestrup, Frank Møller

    2014-01-01

    The draft genome sequences of two copper-resistant Escherichia coli strains were determined. These had been isolated from copper-fed pigs and contained additional putative operons conferring copper and other metal and metalloid resistances.......The draft genome sequences of two copper-resistant Escherichia coli strains were determined. These had been isolated from copper-fed pigs and contained additional putative operons conferring copper and other metal and metalloid resistances....

  1. Convective heat flow probe

    Science.gov (United States)

    Dunn, James C.; Hardee, Harry C.; Striker, Richard P.

    1985-01-01

    A convective heat flow probe device is provided which measures heat flow and fluid flow magnitude in the formation surrounding a borehole. The probe comprises an elongate housing adapted to be lowered down into the borehole; a plurality of heaters extending along the probe for heating the formation surrounding the borehole; a plurality of temperature sensors arranged around the periphery of the probe for measuring the temperature of the surrounding formation after heating thereof by the heater elements. The temperature sensors and heater elements are mounted in a plurality of separate heater pads which are supported by the housing and which are adapted to be radially expanded into firm engagement with the walls of the borehole. The heat supplied by the heater elements and the temperatures measured by the temperature sensors are monitored and used in providing the desired measurements. The outer peripheral surfaces of the heater pads are configured as segments of a cylinder and form a full cylinder when taken together. A plurality of temperature sensors are located on each pad so as to extend along the length and across the width thereof, with a heating element being located in each pad beneath the temperature sensors. An expansion mechanism driven by a clamping motor provides expansion and retraction of the heater pads and expandable packer-type seals are provided along the probe above and below the heater pads.

  2. Biomarkers of waterborne copper exposure in the guppy Poecilia vivipara acclimated to salt water

    Energy Technology Data Exchange (ETDEWEB)

    Machado, Anderson Abel de Souza [Universidade Federal do Rio Grande, Programa de Pós-Graduação em Oceanografia Biológica, Av. Itália km 8, 96201-900 Rio Grande, Rio Grande do Sul (Brazil); Hoff, Mariana Leivas Müller [Universidade Federal do Rio Grande, Instituto de Ciências Biológicas, Av. Itália km 8, 96201-900 Rio Grande, Rio Grande do Sul (Brazil); Klein, Roberta Daniele [Universidade Federal do Rio Grande, Programa de Pós-Graduação em Ciências Fisiológicas – Fisiologia Animal Comparada, Av. Itália km 8, 96201-900 Rio Grande, Rio Grande do Sul (Brazil); Cardozo, Janaina Goulart [Universidade Federal do Rio Grande, Instituto de Ciências Biológicas, Av. Itália km 8, 96201-900 Rio Grande, Rio Grande do Sul (Brazil); Giacomin, Marina Mussoi [Universidade Federal do Rio Grande, Programa de Pós-Graduação em Ciências Fisiológicas – Fisiologia Animal Comparada, Av. Itália km 8, 96201-900 Rio Grande, Rio Grande do Sul (Brazil); Pinho, Grasiela Lopes Leães [Universidade Federal do Rio Grande, Instituto de Oceanografia, Av. Itália km 8, 96201-900 Rio Grande, Rio Grande do Sul (Brazil); and others

    2013-08-15

    Highlights: •Acute effects of waterborne copper were evaluated in the estuarine guppy Poecilia vivipara. •Fishes were acutely exposed to waterborne copper in salt water. •Waterborne copper affects the response of several biochemical and genetic endpoints. •Catalase, reactive oxygen species, antioxidant capacity and lipid peroxidation are responsive to copper exposure. •Copper exposure induces DNA damages in fish erythrocytes. -- Abstract: The responses of a large suite of biochemical and genetic parameters were evaluated in tissues (liver, gills, muscle and erythrocytes) of the estuarine guppy Poecilia vivipara exposed to waterborne copper in salt water (salinity 24 ppt). Activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase, and glutathione S-transferase), metallothionein-like protein concentration, reactive oxygen species (ROS) content, antioxidant capacity against peroxyl radicals (ACAP), and lipid peroxidation (LPO) were evaluated in liver, gills, and muscle. Comet assay score and nuclear abnormalities and micronucleated cell frequency were analyzed in peripheral erythrocytes. The responses of these parameters were evaluated in fish exposed (96 h) to environmentally relevant copper concentrations (5, 9 and 20 μg L{sup −1}). In control and copper-exposed fish, no mortality was observed over the experimental period. Almost all biochemical and genetic parameters proved to be affected by waterborne copper exposure. However, the response of catalase activity in liver, ROS, ACAP and LPO in muscle, gills and liver, and DNA damages in erythrocytes clearly showed to be dependent on copper concentration in salt water. Therefore, the use of these parameters could be of relevance in the scope of biomonitoring programs in salt water environments contaminated with copper.

  3. Multi-Copper Oxidases and Human Iron Metabolism

    Science.gov (United States)

    Vashchenko, Ganna; MacGillivray, Ross T. A.

    2013-01-01

    Multi-copper oxidases (MCOs) are a small group of enzymes that oxidize their substrate with the concomitant reduction of dioxygen to two water molecules. Generally, multi-copper oxidases are promiscuous with regards to their reducing substrates and are capable of performing various functions in different species. To date, three multi-copper oxidases have been detected in humans—ceruloplasmin, hephaestin and zyklopen. Each of these enzymes has a high specificity towards iron with the resulting ferroxidase activity being associated with ferroportin, the only known iron exporter protein in humans. Ferroportin exports iron as Fe2+, but transferrin, the major iron transporter protein of blood, can bind only Fe3+ effectively. Iron oxidation in enterocytes is mediated mainly by hephaestin thus allowing dietary iron to enter the bloodstream. Zyklopen is involved in iron efflux from placental trophoblasts during iron transfer from mother to fetus. Release of iron from the liver relies on ferroportin and the ferroxidase activity of ceruloplasmin which is found in blood in a soluble form. Ceruloplasmin, hephaestin and zyklopen show distinctive expression patterns and have unique mechanisms for regulating their expression. These features of human multi-copper ferroxidases can serve as a basis for the precise control of iron efflux in different tissues. In this manuscript, we review the biochemical and biological properties of the three human MCOs and discuss their potential roles in human iron homeostasis. PMID:23807651

  4. Regulation of copper and iron homeostasis by metal chelators: a possible chemotherapy for Alzheimer's disease.

    Science.gov (United States)

    Robert, Anne; Liu, Yan; Nguyen, Michel; Meunier, Bernard

    2015-05-19

    With the increase of life expectancy of humans in more than two-thirds of the countries in the World, aging diseases are becoming the frontline health problems. Alzheimer's disease (AD) is now one of the major challenges in drug discovery, since, with the exception of memantine in 2003, all clinical trials with drug candidates failed over the past decade. If we consider that the loss of neurons is due to a high level of oxidative stress produced by nonregulated redox active metal ions like copper linked to amyloids of different sizes, regulation of metal homeostasis is a key target. The difficulty for large copper-carrier proteins to directly extract copper ions from metalated amyloids might be considered as being at the origin of the rupture of the copper homeostasis regulation in AD brains. So, there is an urgent need for new specific metal chelators that should be able to regulate the homeostasis of metal ions, specially copper and iron, in AD brains. As a consequence of that concept, chelators promoting metal excretion from brain are not desired. One should favor ligands able to extract copper ions from sinks (amyloids being the major one) and to transfer these redox-active metal ions to copper-carrier proteins or copper-containing enzymes. Obviously, the affinity of these chelators for the metal ion should not be a sufficient criterion, but the metal specificity and the ability of the chelators to release the metal under specific biological conditions should be considered. Such an approach is still largely unexplored. The requirements for the chelators are very high (ability to cross the brain-blood barrier, lack of toxicity, etc.), few chemical series were proposed, and, among them, biochemical or biological data are scarce. As a matter of fact, the bioinorganic pharmacology of AD represents less than 1% of all articles dedicated to AD drug research. The major part of these articles deals with an old and rather toxic drug, clioquinol and related analogs, that

  5. Multispectral imaging probe

    Science.gov (United States)

    Sandison, David R.; Platzbecker, Mark R.; Descour, Michael R.; Armour, David L.; Craig, Marcus J.; Richards-Kortum, Rebecca

    1999-01-01

    A multispectral imaging probe delivers a range of wavelengths of excitation light to a target and collects a range of expressed light wavelengths. The multispectral imaging probe is adapted for mobile use and use in confined spaces, and is sealed against the effects of hostile environments. The multispectral imaging probe comprises a housing that defines a sealed volume that is substantially sealed from the surrounding environment. A beam splitting device mounts within the sealed volume. Excitation light is directed to the beam splitting device, which directs the excitation light to a target. Expressed light from the target reaches the beam splitting device along a path coaxial with the path traveled by the excitation light from the beam splitting device to the target. The beam splitting device directs expressed light to a collection subsystem for delivery to a detector.

  6. NID Copper Sample Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Kouzes, Richard T.; Zhu, Zihua

    2011-02-01

    The current focal point of the nuclear physics program at PNNL is the MAJORANA DEMONSTRATOR, and the follow-on Tonne-Scale experiment, a large array of ultra-low background high-purity germanium detectors, enriched in 76Ge, designed to search for zero-neutrino double-beta decay (0νββ). This experiment requires the use of germanium isotopically enriched in 76Ge. The DEMONSTRATOR will utilize 76Ge from Russia, but for the Tonne-Scale experiment it is hoped that an alternate technology under development at Nonlinear Ion Dynamics (NID) will be a viable, US-based, lower-cost source of separated material. Samples of separated material from NID require analysis to determine the isotopic distribution and impurities. The MAJORANA DEMONSTRATOR is a DOE and NSF funded project with a major science impact. DOE is funding NID through an SBIR grant for development of their separation technology for application to the Tonne-Scale experiment. The Environmental Molecular Sciences facility (EMSL), a DOE user facility at PNNL, has the required mass spectroscopy instruments for making these isotopic measurements that are essential to the quality assurance for the MAJORANA DEMONSTRATOR and for the development of the future separation technology required for the Tonne-Scale experiment. A sample of isotopically separated copper was provided by NID to PNNL for isotopic analysis as a test of the NID technology. The results of that analysis are reported here.

  7. Imaging probe for tumor malignancy

    Science.gov (United States)

    Tanaka, Shotaro; Kizaka-Kondoh, Shinae; Hiraoka, Hasahiro

    2009-02-01

    Solid tumors possess unique microenvironments that are exposed to chronic hypoxic conditions ("tumor hypoxia"). Although more than half a century has passed since it was suggested that tumor hypoxia correlated with poor treatment outcomes and contributed to cancer recurrence, a fundamental solution to this problem has yet to be found. Hypoxia-inducible factor (HIF-1) is the main transcription factor that regulates the cellular response to hypoxia. It induces various genes whose functions are strongly associated with malignant alteration of the entire tumor. The cellular changes induced by HIF-1 are extremely important targets of cancer therapy, particularly in therapy against refractory cancers. Imaging of the HIF-1-active microenvironment is therefore important for cancer therapy. To image HIF-1activity in vivo, we developed a PTD-ODD fusion protein, POHA, which was uniquely labeled with near-infrared fluorescent dye at the C-terminal. POHA has two functional domains: protein transduction domain (PTD) and VHL-mediated protein destruction motif in oxygen-dependent degradation (ODD) domain of the alpha subunit of HIF-1 (HIF-1α). It can therefore be delivered to the entire body and remain stabilized in the HIF-1-active cells. When it was intravenously injected into tumor-bearing mice, a tumor-specific fluorescence signal was detected in the tumor 6 h after the injection. These results suggest that POHA can be used an imaging probe for tumor malignancy.

  8. Zinc Supplementation Overcomes Effects of Copper on Zinc Status, Carbohydrate Metabolism and Some Enzyme Activities in Diabetic and Nondiabetic Rats.

    Science.gov (United States)

    Derouiche, Samir; Kechrid, Zine

    2016-08-01

    The aim of this study was to investigate the effect of zinc supplementation on zinc status, carbohydrate metabolism and some enzyme activities in rats with alloxan-induced diabetes that were fed high-copper feed. Male albino Wistar rats were randomly divided into 6 groups (n=10). The first and fourth groups were nondiabetic and diabetic controls. The second, third, fifth and sixth groups were copper, copper + zinc, diabetes + copper and diabetes + copper + zinc groups, respectively. Diabetes in the fourth, fifth and sixth groups was induced by alloxan. Copper (30 mg/kg feed) as CuSO4 5H2O and zinc (231 mg/kg feed) as ZnSO4 7H2O were added to the feed of the animals in the copper and zinc groups for 21 days. Copper supplementation caused a significant decrease in body weight gain, serum zinc, tissue zinc, serum protein concentrations, alkaline phosphatase, lactic dehydrogenase and amylase activities. In contrast, it led to an augmentation in creatinine, uric acid and transaminases activities in rats with and without diabetes. Zinc supplementation in the feed for animals given copper ensured a partial correction of the previous parameters. The study demonstrated the beneficial effects of zinc treatment in copper-induced metabolic disturbance in diabetic and nondiabetic rats. Copyright © 2016 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.

  9. Sub-cellular damage by copper in the cnidarian Zoanthus robustus.

    Science.gov (United States)

    Grant, A; Trompf, K; Seung, D; Nivison-Smith, L; Bowcock, H; Kresse, H; Holmes, S; Radford, J; Morrow, P

    2010-09-01

    Sessile organisms may experience chronic exposure to copper that is released into the marine environment from antifoulants and stormwater runoff. We have identified the site of damage caused by copper to the symbiotic cnidarian, Zoanthus robustus (Anthozoa, Hexacorallia). External changes to the zoanthids were apparent when compared with controls. The normally flexible bodies contracted and became rigid. Histological examination of the zoanthid tissue revealed that copper had caused sub-cellular changes to proteins within the extracellular matrix (ECM) of the tubular body. Collagen in the ECM and the internal septa increased in thickness to five and seven times that of controls respectively. The epithelium, which stained for elastin, was also twice as thick and tough to cut, but exposure to copper did not change the total amount of desmosine which is found only in elastin. We conclude that copper stimulated collagen synthesis in the ECM and also caused cross-linking of existing proteins. However, there was no expulsion of the symbiotic algae (Symbiodinium sp.) and no effect on algal pigments or respiration (44, 66 and 110 microg Cu L(-1)). A decrease in net photosynthesis was observed only at the highest copper concentration (156 microg Cu L(-1)). These results show that cnidarians may be more susceptible to damage by copper than their symbiotic algae. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  10. A copper-transporting ATPase BcCCC2 is necessary for pathogenicity of Botrytis cinerea.

    Science.gov (United States)

    Saitoh, Yoshimoto; Izumitsu, Kosuke; Morita, Atsushi; Tanaka, Chihiro

    2010-07-01

    Copper is an essential trace element that serves as a cofactor for numerous enzymes. In eukaryotes, copper-transporting ATPases deliver copper to various copper-containing proteins in the trans-golgi network. This study identified a copper-transporting ATPase gene BcCcc2 in a fungus pathogenic to plants, Botrytis cinerea. We investigated the biological roles of BcCCC2 by generating null mutants for BcCcc2. Melanization, conidiation and the formation of sclerotia were severely affected in DeltaBcCcc2 mutants. Moreover, a pathogenicity assay using tomato leaves and carnation petals revealed the mutants to be nonpathogenic. Further analysis indicated that they formed fewer appressoria and infection cushions than the wild-type. These structures were aberrant in morphology and in many cases had a significantly reduced ability to penetrate the plant epidermis. An assay also indicated that DeltaBcCcc2 mutants were defective in infection through wounds. BcCCC2 is necessary not only for penetrating a host but also for fungal growth within plant tissues. Our results also imply that B. cinerea requires copper-containing proteins for infection that are inactive in the absence of the copper-transporting ATPase BcCCC2.

  11. Carnitine supplementation modulates high dietary copper-induced oxidative toxicity and reduced performance in laying hens.

    Science.gov (United States)

    Güçlü, Berrin Kocaoğlu; Kara, Kanber; Çakır, Latife; Çetin, Ebru; Kanbur, Murat

    2011-12-01

    This experiment was conducted to evaluate the effects of L-carnitine on performance, egg quality and certain biochemical parameters in laying hens fed a diet containing high levels of copper proteinate. Forty-eight 42-week-old laying hen